Application of circular RNA molecule circ-PHF21A in preparation of glioma diagnostic kit and drug, and glioma diagnostic kit and drug

Abstract

The invention provides application of a circular RNA molecule circ-PHF21A in preparation of a diagnostic kit and a drug for glioblastoma, and the diagnostic kit and the drug of the circular RNA molecule circ-PHF21A. According to the application disclosed by the invention, low expression of circ-PHF21A in a glioblastoma tissue is found through a bioinformatics method, and malignant biological behaviors of glioblastoma are inhibited through overexpression of circ-PHF21A. Meanwhile, the circular RNA is found to be capable of coding functional protein so as to play a tumor inhibition role. Therefore, the circ-PHF21A can be used as a molecular marker for malignant progression of the glioblastoma, and a new diagnosis and treatment strategy is provided for treatment of the glioblastoma.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and oncology, and specifically relates to the application of a circular RNA molecule circ-PHF21A in glioma. Background Art

[0002] Glioma is the most common primary central nervous system tumor. Current studies believe that it originates from glial stem cells or progenitor cells. Among them, glioblastoma (GBM), as a WHO grade IV glioma, is highly invasive and accounts for approximately 51.5% of all malignant brain tumors. According to current diagnosis and treatment standards, the standard treatment for GBM is maximum surgical resection supplemented with radiotherapy and temozolomide (TMZ) chemotherapy. However, the clinical prognosis is still not ideal, with the median overall survival of patients being only 14.6-20.5 months, and the recurrence rate being as high as over 90%.

[0003] It is worth noting that, with the exception of MGMT promoter methylation status, which can be used as a predictor of TMZ chemotherapy sensitivity, existing phase III clinical trial data show that molecular typing based on genomic and transcriptomic characteristics has no significant impact on the selection of existing treatment options and the prediction of efficacy. Even more regrettable is that to date, no molecular targeted drugs or immunotherapy drugs have achieved breakthrough progress in the treatment of GBM. The main reason for this is related to GBM's unique biological characteristics, such as strong invasiveness, high heterogeneity, immune evasion, blood-brain barrier, and treatment resistance. These factors have led to major challenges in GBM treatment and highlighted the urgent need to develop new treatment strategies.

[0004] Circular RNA (circRNA) is a covalently closed loop transcript lacking the typical 5' cap and 3' polyadenylated tail of linear RNA. It is widely found in eukaryotes and is highly stable. It is generally considered to be a non-coding RNA produced by transcription errors or byproducts. With the rapid development of whole-genome sequencing and translatomics sequencing technologies, circRNAs have been linked to a variety of human diseases, including diabetes, neurological diseases, and cardiovascular diseases. In recent years, a large number of new circRNA-related studies have emerged in the field of oncology. For example, circ-0079593 promotes the malignant progression of glioma by sponging miR-324-5p. Compared with linear mRNA, circRNAs have greater stability and lower immunogenicity, which makes them promising for broad application in biomarker discovery, vaccine development, tumor immunotherapy, and protein replacement therapy.

[0005] Although conventional surgery and chemoradiotherapy have made some progress in the treatment of GBM, current approaches remain limited by their lack of targeting, tumor heterogeneity, and the blood-brain barrier. Therefore, screening for novel molecular diagnostic markers and developing precise targeted therapeutic strategies have become key research breakthroughs. Summary of the Invention

[0006] In response to the above-mentioned problems, one of the objectives of the present invention is to provide the use of the circular RNA molecule circ-PHF21A in the detection and treatment of GBM.

[0007] A second object of the present invention is to provide a kit prepared from the circular RNA molecule circ-PHF21A and a pharmaceutical composition containing the circular RNA molecule circ-PHF21A.

[0008] The present invention specifically provides the following technical solutions:

[0009] 1. Use of the circular RNA molecule circ-PHF21A in preparing a GBM diagnostic kit, wherein the CircBase ID of the circ-PHF21A is hsa_circ_0000296, and the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO: 1. The structure of the circular RNA molecule circ-PHF21A is a circular structure formed by connecting the nucleotide sequence shown in SEQ ID NO: 1 end to end.

[0010] 2. Use of the circular RNA molecule circ-PHF21A in the preparation of a drug for the treatment of GBM. The CircBase ID of the circ-PHF21A is hsa_circ_0000296, and the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO: 1. The structure of the circular RNA molecule circ-PHF21A is a circular structure formed by connecting the nucleotide sequence shown in SEQ ID NO: 1 end to end.

[0011] 3. A GBM diagnostic kit, comprising primers capable of amplifying a circular RNA molecule, circ-PHF21A, whose nucleotide sequence is shown in SEQ ID NO: 1. The amplification primers are a primer pair consisting of the DNA sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, or a primer pair consisting of the DNA sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0012] 4. A pharmaceutical composition comprising a circular RNA molecule circ-PHF21A, wherein the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO: 1.

[0013] 5. A polypeptide characterized by its amino acid sequence as shown in SEQ ID NO: 17. The polypeptide is used in the preparation of a glioblastoma diagnostic kit or a drug for treating glioblastoma. The polypeptide, named "PHF21A-74aa," is encoded by circ-PHF21A and is 74 amino acids long.

[0014] The relevant contents of the above technical solution are explained as follows:

[0015] The technologies involved in the present invention are all conventional molecular cloning techniques. The enzymes, primers, reagents and reaction conditions involved can be reasonably selected based on the experience of those skilled in the art unless otherwise specified. The reagents and consumables involved are common products available on the market, and the detection methods and instruments involved are also well known and proficient in by those skilled in the art.

[0016] Initially, the inventors collected 10 pairs of fresh GBM tissue specimens and paired adjacent brain tissue specimens. High-throughput sequencing analysis was performed using circular RNA sequencing and ribosomal imprint sequencing (Ribo-seq). Bioinformatics analysis screened for differentially expressed circRNAs between GBM and adjacent tissues, ultimately identifying 25,231 significantly differentially expressed circRNAs. By combining circRNA-seq, Ribo-seq, peptide quantification data, circRNA ORF annotation, and IRES finder software analysis, circRNAs derived from the same peptide were removed, ultimately identifying 11 candidate circRNAs. Combining relevant literature with differential expression data, the significantly differentially expressed circ-PHF21A was selected. Based on circBase data, circ-PHF21A (hsa_circ_0000296) is a 277-nucleotide circRNA formed by back-splicing of exons 2, 3, 4, and 5 of the PHF21A gene on human chromosome 11. We identified circ-PHF21A as encoding a 74-amino acid polypeptide, which was named “PHF21A-74aa” (SEQ ID NO: 17).

[0017] Next, the inventors constructed a plasmid overexpressing circ-PHF21A and tagged with a Flag tag and transfected it into 293T cells. Western blotting (WB) confirmed the presence of PHF21A-74aa encoded by circ-PHF21A in 293T cells. Circ-PHF21A was overexpressed in GBM cell lines, and CCK-8, EdU staining, cell wound healing, and Transwell invasion assays revealed for the first time the inhibitory effects of circ-PHF21A on GBM cell proliferation, migration, and invasion.

[0018] The technical solution of the present invention has the following advantages and effects:

[0019] (1) The present invention confirmed the circular stability of circ-PHF21A through second-generation high-throughput sequencing and RNase R degradation experiments;

[0020] (2) The present invention analyzed sequencing data through bioinformatics and found that the expression level of circ-PHF21A was significantly reduced in GBM, and therefore, it can be used as a potential diagnostic marker for GBM;

[0021] (3) The present invention conducted an in vitro cellular functional study on circ-PHF21A circular RNA. By cloning the sequence of circ-PHF21A into the LVminiCirc lentiviral vector, GBM cell lines (U87, U251) that stably overexpressed circ-PHF21A were successfully constructed. The experimental results showed that compared with the control group, overexpression of circ-PHF21A significantly inhibited the proliferation, migration and invasion of GBM cells, suggesting that it may play a tumor suppressor role in the progression of GBM. The present invention reveals the tumor suppressor function of circ-PHF21A in GBM. circ-PHF21A and its expression products can be used as diagnostic markers for GBM, which helps to improve the accuracy and efficiency of diagnosis, and as potential target genes for the treatment of GBM, it provides new therapeutic strategies and targets for drug development.

[0022] In general, there are currently no reports on the function of circ-PHF21A. The present inventors discovered the cyclization phenomenon of the PHF21A gene in their research, and through bioinformatics methods, they found that circ-PHF21A was significantly underexpressed in GBM tissue. They also found that this circRNA can encode functional proteins, thereby exerting a tumor-suppressing effect, which was verified by cell functional experiments. By overexpressing circ-PHF21A, it was found that the malignant biological behavior of GBM cells was inhibited. The present invention reveals that circ-PHF21A can be used as a molecular marker for the malignant progression and diagnosis and treatment of GBM, providing new ideas for the diagnosis and targeted treatment of GBM, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attachment Figure 1 This is the map of the pLC5-ciR plasmid vector.

[0024] Attachment Figure 2 The detection graph of circ-PHF21A expression levels in 10 pairs of GBM samples and corresponding adjacent cancer tissues.

[0025] Attachment Figure 3 The results of RT-qPCR and agarose gel electrophoresis (using divergent primers and convergent primers to amplify circ-PHF21A in the templates of GBM cell lines U87 and U251, respectively).

[0026] Attachment Figure 4 Figure 2 shows the CCK-8 experimental results after U251 cells overexpressed circ-PHF21A.

[0027] Attachment Figure 5 Fluorescence microscopy image of the constructed cell line stably expressing circ-PHF21A (GFP positivity > 70%).

[0028] Attachment Figure 6 This is the result of EdU staining experiment after U251 cells overexpressed circ-PHF21A.

[0029] Attachment Figure 7 The results of the scratch healing experiment after U251 cells overexpressed circ-PHF21A.

[0030] Attachment Figure 8 Figure 2 shows the results of Transwell invasion assay of U87 and U251 cells after overexpressing circ-PHF21A.

[0031] Attachment Figure 9 This figure shows the results of WB experiment to detect the expression of peptide PHF21A-74aa after overexpressing circ-PHF21A in 293T cells. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0033] The molecular biology experimental techniques used in the following examples include vector construction, PCR amplification, plasmid transfection, CCK-8, EdU staining, cell scratching, Transwell invasion, WB, etc. Unless otherwise specified, they were generally performed according to conventional methods.

[0034] 1. Screening of GBM-related circRNA molecules.

[0035] 1. Sequencing sample collection

[0036] This study included 10 patients with GBM who underwent surgery and were admitted to the Department of Neurosurgery at Beijing Tiantan Hospital between July and September 2021. Patients with a confirmed diagnosis by postoperative pathology were included. Patients who had received adjuvant chemoradiotherapy, recurrent GBM, or underwent tumor biopsy alone were strictly excluded. All tissue samples were obtained intraoperatively, quickly frozen in liquid nitrogen, and uniformly transferred to a -80°C ultra-low temperature freezer for classified cryopreservation. This study was approved by the Ethics Committee of the Department of Neurosurgery at Beijing Tiantan Hospital, and all patients signed informed consent forms for the use of surgically resected specimens for clinical research before surgery.

[0037] 2. RNA extraction, library construction and sequencing

[0038] Total RNA was extracted using a Trizol kit according to the manufacturer's instructions. RNA quality was assessed using an Agilent 2100 Bioanalyzer and verified by RNase-free agarose gel electrophoresis. Extracted total RNA was treated with RNase R to degrade linear RNA and purified using an RNeasy MinElute cleanup kit. Strand-specific libraries were constructed for Illumina using the VAHTS Total RNAseq (H / M / R) Library Prep Kit according to the manufacturer's instructions. Ribosomal RNA was removed to preserve circular RNA. The enriched circular RNA was treated with fragmentation buffer to break it into short fragments and reverse transcribed into cDNA using random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTPs (dUTP was used instead of dTTP), and buffer. The cDNA fragments were purified using VAHTS™ DNA Clean Beads, end-repaired, A-tailed, and ligated to Illumina sequencing adapters. Subsequently, the second-strand cDNA was digested with UNG. The digestion products were purified using VAHTS™ DNA Clean Beads, amplified by PCR, and sequenced using the Illumina Novaseq60000. The high-throughput sequencing in this study was provided by Guangzhou Kidio, which provided all the instruments, reagents, and data analysis solutions.

[0039] 3. Bioinformatics analysis flow chart

[0040] 3.1 Raw data filtering

[0041] The raw reads obtained from the sequencer contain adapters or low-quality bases, which can affect subsequent analysis. Therefore, to obtain high-quality clean reads, fastp (version 0.18.0) was used for further filtering. The parameters are as follows:

[0042] (1) remove reads containing adapters;

[0043] (2) reads containing more than 10% unknown bases (N) were removed;

[0044] (3) Removal of low-quality reads: reads containing more than 50% low-quality (Q value ≤ 20) bases were removed.

[0045] 3.2 Ribosomal RNA (rRNA) alignment

[0046] Different species and sample quality can affect the efficiency of ribosomal RNA removal in experiments. Therefore, reads were aligned to a ribosomal RNA (rRNA) database using the short sequence alignment tool Bowtie2 (version 2.2.8). Reads matching rRNA were removed. The remaining reads were used for further alignment and analysis.

[0047] 3.3 Alignment with the reference genome

[0048] After removing rRNA from each sample, reads were mapped to the reference genome using TopHat2 (version 2.1.1). Reads that successfully aligned to the reference genome were discarded, and reads that failed to map to the genome were collected for circRNA identification.

[0049] 3.4 Identification of circRNAs

[0050] 20 nucleotides were extracted from both ends of unmapped reads and aligned to the reference genome to find unique anchor sites for splice sites. Anchored reads that docked in the reverse direction (head-to-tail) indicated circRNA splicing, and then circRNAs were identified using find_circ (version 1). After anchor alignment extension, the complete reads were docked to the GU / AG splice sites of the splice breakpoints. Candidate circRNAs were considered circRNAs if they were supported by at least two unique reverse-splicing reads in at least one sample.

[0051] 3.5 circRNA Statistics

[0052] The types, chromosome distribution and length distribution of the identified circRNAs were statistically analyzed.

[0053] 3.6 Functional enrichment analysis of source genes

[0054] Source genes are the source genes of circRNAs. Functional enrichment analysis was performed on the source genes to investigate their main functions.

[0055] 3.6.1GO enrichment analysis

[0056] Gene Ontology (GO) is an internationally standardized gene function classification system that provides dynamically updated controlled vocabulary and strictly defined concepts to comprehensively describe the properties of genes and their products in any organism. GO has three ontologies: molecular function, cellular component, and biological process. GO enrichment analysis provides GO terms that are significantly enriched in source genes compared with the genomic background, and screens out source genes with corresponding biological functions. First, all source genes are mapped to GO terms in the GO database (http: / / www.geneontology.org / ), the number of genes for each term is calculated, and the hypergeometric test is used to screen out significantly enriched GO terms. The calculation formula is as follows:

[0057]

[0058] N is the total number of genes with GO annotations; n is the number of source genes in N; M is the total number of genes annotated to a particular GO term; and m is the number of source genes in M. The calculated P value was FDR-corrected, with an FDR ≤ 0.05 as the threshold. GO terms that met this condition were defined as significantly enriched in the source genes. This analysis can identify the primary biological functions exerted by the source genes.

[0059] 3.6.2 Pathway enrichment analysis

[0060] Genes often play a role in certain biological functions through interactions. Pathway-based analysis helps further understand the biological functions of genes. KEGG is a major public pathway-related database. Pathway enrichment analysis identifies metabolic pathways or signal transduction pathways that are significantly enriched in the source genes by comparing them to the whole genome context. The calculation formula is the same as that used in GO analysis:

[0061]

[0062] Where N is the total number of genes with KEGG annotations; n is the number of source genes in N; M is the total number of genes annotated to a specific pathway; and m is the number of source genes in M. The calculated P value was FDR-corrected, with an FDR ≤ 0.05 as the threshold. Pathways meeting this condition were defined as pathways significantly enriched in the source genes.

[0063] 3.7 Quantification of circRNA Abundance

[0064] To quantify circRNAs, the number of reads at the backsplicing junctions was normalized to reads per million mapped (RPM) using the following formula:

[0065]

[0066] C is the number of reads uniquely aligned to the backsplicing junction of a circRNA; N is the total number of reads at the backsplicing junction. The RPM method eliminates the impact of varying sequencing data volumes on circRNA expression calculations. Therefore, the calculated expression can be directly used to compare differential expression between different samples.

[0067] 3.8 Analysis of differentially expressed circRNAs

[0068] To identify differentially expressed circRNAs between different samples or groups, the edgeR package (version 3.12.1) (http: / / www.r-project.org / ) was used for analysis. We compared between samples or groups and screened circRNAs with log2fold change ≥ 1 and P value < 0.05 as significantly differentially expressed circRNAs.

[0069] 3.9 circRNA Database Annotation

[0070] circRNAs were annotated by BLAST comparison against circBase. CircRNAs that could not be annotated were defined as novel circRNAs.

[0071] 3.10 Screening of circRNAs

[0072] Differential expression analysis was used to screen for circRNAs with differential expression between GBM and adjacent tissues (log2 fold change ≥ 1 and P value < 0.05), ultimately yielding 25,231 significantly differentially expressed circRNAs. CircRNA-seq, Ribo-seq, peptide quantification data, circRNA ORF annotation, and IRESfinder software were combined to remove circRNAs with the same peptide source and meeting the following criteria: (1) circRNAs present in the circBase database; (2) their ORFs were expressed in at least one sample in the GBM group, with a total of 3 reads across the interface; and (3) the ORF length across the circRNA interface was shorter than the circRNA length and contained a ribosome entry site. Eleven candidate circRNAs were ultimately identified. Combining relevant literature with differential expression data, circ-PHF21A, which was significantly differentially expressed, was selected.

[0073] (The results are shown in Figure 2 )

[0074] 2. Vector Construction and Identification

[0075] 2.1. Overexpression vector construction and identification

[0076] The full-length 355bp sequence of the hsa_circ_0000296-3xFlag gene was amplified by PCR and ligated into the pLC5-ciR vector using In-Fusion cloning technology.

[0077] PCR primers are as follows:

[0078] 296-3xFlag-UnF:

[0079] CATTAATATTTCTCTTTCGAATTCTAATACTTTCAGGGGGCTAGAGAGCTGAAGGAG

[0080] 296-3xFlag-UnR:

[0081] AGTATGGAGTTGTTAGCTAGGATCCAGTTGTTCTTACCTGTTTCTCACTCAAAGCTGT

[0082] Vector map: The pLC5-ciR vector was successfully constructed and inserted into the hsa_circ_0000296-3xFlag gene. The insertion sequence was confirmed to be accurate by sequencing. Figure 1 .

[0083] Translation product: Expected ORF (100 aa, 11.48 kDa) expression product. 3xFlag tag was used to label the target protein.

[0084] T2 construction vector ZX220722: hsa_circ_0000296-3xFlag circular overexpression vector

[0085] >hsa_circ_0000296-3xFlag|355nt(pLC5-ciR)

[0086] ForwardCircularFrame—

[0087] GGGGCTAGAGAGCTGAAGGAGCCAGTTTCCCCAAAATTGCTGCAGTGAGAAGAGGAGTTTGTTACTTggtggttctggtGACTACAAAGACCATGACGGTGATTATAAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAAACAGAGGCTGAAGAAACTATAGAATTA GCAGAGAAAGTGGAGAAGGTAGAGGATGGAGTTGCAGACTCTACAGGAGGCTCTTAAAGTGGAAATTCAGGTTTCACCAGAAACTGGTTGCTCAAATGAAGCAGGATCCACAGAATGCTGACTTAAAGAAACAGCTTCATGAACTCCAAGCCAAAATCACAGCTTTGAGTGAGAAACAG

[0088] —Backward Circular Frame

[0089] >3xFlag

[0090] GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAG

[0091] translation=DYKDHDGDYKDHDIDYKDDDDK

[0092] Linker GGSG:ggtggttctggt

[0093] Translation product: >ORF (100aa, 11.48kDa)

[0094] MELQTLQEALKVEIQVHQKLVAQMKQDPQNADLKKQLHELQAKITALSEKQGARELKESQFPQNCCSEKRSLLLGGSGDYKDHDGDYKDHDIDYKDDDDK.

[0095] The sequencing peak graph was normal, without any mixed peaks or overlapping bands, and the sequence alignment was consistent, indicating that hsa_circ_0000296-3xFlag was successfully inserted into pLC5-ciR and the overexpression vector was successfully constructed.

[0096] Vector sequencing primer: pC5-seqR:TCCTCTCTTGATTTCCTTATT

[0097] 2.2 Cell transfection

[0098] The constructed overexpression vector (hsa_circ_0000296-3xFlag) and the control group (pLC5-ciR) were transfected into 293T cells, respectively. 48 hours after transfection, the cells were collected for qPCR detection to verify the overexpression efficiency.

[0099] 2.3 qPCR verification of expression efficiency after transfection

[0100] Total RNA was extracted from cell samples and subjected to qPCR after reverse transcription. GAPDH was used as an internal control for data normalization, and hsa_circ_0000296-3xFlag specific primers were used for detection.

[0101] 2.4 qPCR detection

[0102] 2.4.1 RNA extraction

[0103] 2.4.1.1 Sample processing

[0104] (1) Add 1 ml of Trizol to a mung bean-sized piece of tissue, homogenize it using a Tissue Ruptor for 1 minute, and let it stand at room temperature for 10 minutes;

[0105] (2) Add 200 μl of chloroform, mix thoroughly, and centrifuge at 15,000 rpm for 5-7 minutes.

[0106] (3) Take the supernatant, transfer it to a 1.5 ml Eppendorf tube, add 600 μl of chloroform, mix well, and centrifuge at 15,000 rpm for 5 minutes.

[0107] (4) Take the supernatant, transfer it to a 1.5ml Eppendorf tube, add 500ul of isopropanol, mix well, and centrifuge at 15,000 rpm for 10 minutes.

[0108] (5) Discard the supernatant, rinse with 1 ml of 75% ethanol, and centrifuge at high speed for 5 minutes.

[0109] (6) Discard the supernatant and allow the RNA pellet to air dry (2-3 minutes).

[0110] (7) Dissolve the dried RNA in RNA-free water.

[0111] (8) Take 1 μl of total RNA, measure OD260, and quantify.

[0112] 2.4.2 Reverse transcription

[0113] (1) Add the following reactants to a 200 μl PCR tube (volume 12 μl) in the following order:

[0114] 1.DEPC water (10-x) μl

[0115] 2. Random primers / Oligo dT (50pM / ul) 2μl

[0116] 3. RNA xμl (2ug)

[0117] (1) In a PCR instrument, treat at 65°C for 5 minutes;

[0118] (2) Immediately place in an ice bath and centrifuge at high speed (above 5000 g) for 5 seconds;

[0119] (3) Add the following reactants to the PCR tube in the following order (total volume after addition is 20 μl):

[0120]

[0121] (4) After mixing, incubate at 25°C for 5 minutes and then at 42°C for 60 minutes. (For samples with high GC content, the temperature can be increased to 45°C.)

[0122] (5) 70°C for 5 minutes;

[0123] (6) Centrifuge at high speed (greater than 5000g) for 5 seconds. Store at -20°C. If longer storage is required, place in a -80°C freezer.

[0124] 2.4.3 Fluorescence quantitative PCR amplification

[0125] (1) Sequence and primer design,

[0126] The sequences were referenced to the sequences of each target gene in the Gene Bank database, and the primers were designed by NCBI Primer-blast.

[0127] qPCR primers:

[0128] GAPDH-F:AGAAGGCTGGGGCTCATTTG

[0129] GAPDH-R:GCAGGAGGCATTGCTGATGAT

[0130] hsa_circ_0000296-3xFlag-F2:TCACCAGAAACTGGTTGCTC

[0131] hsa_circ_0000296-3xFlag-R2:AGCTCTCTAGCCCCCTGTT

[0132] Amplified fragment size: 120bp

[0133] (2) PCR reaction system (10 μl)

[0134] H2O 1.5μl

[0135] 2×SYBGEEN PCR mix 5μl

[0136] Primer (10uM) 1μl

[0137] Template (reverse transcription product, also known as cDNA) 2.5 μl

[0138] (3) Reaction conditions

[0139] 1.95℃ 2 minutes

[0140] 2.95℃ 5 seconds

[0141] 3.60℃ 10 seconds 45cycles

[0142] 4. Melt Curve

[0143] 2.4.4 qPCR Results Analysis

[0144] Relative expression differences were calculated using the 2-ΔΔCT method. hsa_circ_0000296-3×Flag showed significantly higher expression (167-fold) than the control group after transfection into 293T cells. Sequencing of the PCR product fully matched the reference sequence of the circularization site, indicating that the constructed overexpression vector successfully expressed the target circRNA.

[0145] The cyclization site sequence of hsa_circ_0000296-3xFlag in circBase is TGAGAAACAGGGGGCTAGA

[0146] qPCR product sequencing primers:

[0147] hsa_circ_0000296-3xFlag-F2:TCACCAGAAACTGGTTGCTC

[0148] hsa_circ_0000296-3xFlag-R1:ACCTTCTCCACTTTCTCTGC

[0149] Amplified fragment size: 303bp

[0150] The above results indicate that hsa_circ_0000296-3xFlag can successfully overexpress the target circRNA in eukaryotic cells.

[0151] 3. Construction of Lentivirus and Its Stable Cell Line

[0152] 1. Cell Seeding

[0153] Take 293T cells in good logarithmic growth phase, digest with trypsin, count and inoculate them into 10 cm cell culture dishes (about 5-6×10 cells per dish). 6 ), cultured overnight in a 37°C, 5% CO2 incubator;

[0154] 2. Plasmid Transfection

[0155] 2.1 Preparation of transfection reagent

[0156] Take 1 ml of pure Opti-MEM medium, take 8 μg of lentiviral packaging plasmid reagent B and expression plasmid and add them to the medium, then add 48 μl of transfection reagent A, shake and mix, and let it stand at room temperature for 30 minutes.

[0157] 2.2 Cell culture medium replacement

[0158] Select 293T cells with a cell growth density of approximately 90%. 30 minutes before plasmid transfection, change the cell culture medium. Gently aspirate the old culture medium and add 10 ml of fresh DMEM complete culture medium.

[0159] 3. Plasmid Transfection

[0160] Add the prepared transfection reagent dropwise to the 293T cells that have been replaced with fresh culture medium, shake evenly, and continue culturing in the incubator.

[0161] 3.1 Collection of viral supernatant

[0162] The culture supernatant was collected 24 hours, 48 ​​hours, and 72 hours after transfection, and fresh culture medium was carefully added after the supernatant was collected at 24 hours and 48 hours to continue the culture.

[0163] 3.2 Centrifugation and filtration of viral supernatant

[0164] The collected viral supernatant was centrifuged at 4°C, 4000 g for 10 minutes, and the viral supernatant was collected; the viral supernatant was filtered through a 0.22 μm filter.

[0165] 3.3 Viral supernatant concentration

[0166] Centrifuge in a 40ml ultracentrifuge tube at 25,000 rpm for 3 hours at 4°C. Discard the supernatant and add 50-100 μl of serum-free medium to resuspend the pellet and collect the concentrated virus. Once fully dissolved, centrifuge at 10,000 rpm for 5 minutes. Aliquot the supernatant and prepare the sample for testing.

[0167] 3.4 Lentivirus quality testing

[0168] The key points of lentivirus quality control include physical status testing and sterility testing.

[0169] 3.4.1 Physical indicator testing

[0170] (1) Color determination: The purified lentivirus appears as a pink clear liquid when visually inspected.

[0171] (2) Viscosity determination: Use a 20-200 μl pipette to slowly aspirate 50 μl of lentivirus. There should be no obvious viscosity or aspiration lag.

[0172] 3.4.2 Sterility testing

[0173] The virus was added to 293T cells for verification. After 24 hours of normal culture, microscopic examination showed no bacterial or fungal contamination. At the same time, referring to the empty cell group, there were no obvious particles in the intercellular space and the culture medium was clear and transparent.

[0174] 3.5 Lentiviral titer determination

[0175] 3.5.1 Fluorescence titer determination

[0176] (1) Take 293T cells in good logarithmic growth phase, digest with trypsin, count and inoculate into 24-well plates (1×10 cells per well). 5 ), culture in a 37°C, 5% CO2 incubator for about 6 hours until the cells adhere;

[0177] (2) Take 5 μl of virus concentrate and dilute it 100-fold with culture medium;

[0178] (3) Add 10ul, 2ul, and 1ul of virus dilution (equivalent to 0.1ul, 0.02ul, and 0.01ul of virus stock solution) to three wells of a 24-well plate, respectively, and continue incubating in an incubator for 72 hours;

[0179] (4) After 72 hours, observe the cell fluorescence in each well and take pictures using a fluorescence microscope;

[0180] (5) Collect cells from each well for flow cytometry and calculate the titer.

[0181] 3.5.2 Drug screening method for titer determination

[0182] Steps 1-3 are the same as for the fluorescence titering method. Add the resistance drug puromycin 72 hours after infection, maintaining the drug concentration at 5 μg / ml. Continue culturing for 1 day and observe cell growth.

[0183] 3.6 Identification of stable cell lines: The established stable cell lines were photographed and observed under a fluorescence microscope. The GFP positive rate was >70%. Figure 5 .

[0184] IV. CCK-8 assay in U251 cells overexpressing circ-PHF21A

[0185] (1) Preparation of cell suspension: Cell counting.

[0186] (2) Inoculation into 96-well plates: Based on the appropriate number of cells to be plated, approximately 100 μl of cell suspension was added to each well, and three replicate wells were set up.

[0187] (3) Cell culture: After inoculation, cells need to be cultured for about 2 to 4 hours to adhere to the wall.

[0188] (4) Add 10 μl of CCK-8 solution to each well. Since the amount of CCK-8 solution added to each well is small, in order to reduce the error caused by the reagent sticking to the well wall or the pipette tip, gently tap the culture plate after adding the reagent to help mix it, or directly prepare a culture medium containing 10% CCK-8 solution and add it in the form of liquid replacement.

[0189] (5) Incubate in the incubator for 2 hours. If the color development is insufficient, continue incubation to confirm the optimal conditions.

[0190] (6) Measure the absorbance at 450 nm. Figure 4 .

[0191] 5. EdU staining experiment of U251 cells overexpressing circ-PHF21A

[0192] 1. Marking:

[0193] Dilute EDU solution (reagent A) with cell culture medium at a ratio of 1000:1 to prepare 1600 μl of EDU medium. Add 100 μl of EDU medium to each well of a 48-well plate and incubate for 3 hours.

[0194] 2. Cell fixation and permeabilization:

[0195] (1) Add 150 μl of 4% paraformaldehyde cell fixative to each well, incubate at room temperature for 30 minutes, and discard the fixative.

[0196] (2) Add 150-200 μl of 2% Triton X-100 permeabilization solution to each well and permeabilize for 15 minutes at room temperature. Discard the permeabilization solution.

[0197] (3) Add 300 μl of PBS to each well and wash twice at room temperature;

[0198] 3. Dyeing:

[0199] (1) Prepare 1X EDU counter buffer by diluting 10X reaction buffer with deionized water in a 10:1 ratio.

[0200] (2) Weigh an appropriate amount of powder and dissolve it in the ratio of 200 mg of buffer additive to 1 ml of deionized water. This is the concentration of the buffer additive that can be used. It is recommended to prepare it before use and dye it for at least half an hour.

[0201] Note: The buffer additive is a white powder, which is difficult to weigh accurately. The weighing range can be slightly relaxed, but should not exceed ±20%. The powder is easily oxidized. Please tighten the tube cap after use. If the reagent appears brown, it needs to be replaced or scrapped.

[0202] (3) Prepare the staining reaction solution according to the following table:

[0203] Staining reaction solution composition 500ul 1ml 2ml 5ml 1X reaction buffer 430ul 860ul 1.8ml 4.3ml Catalyst solution 20ul 40ul 80ul 200ul TAMRA red fluorescent solution 1.2ul 2.5ul 5ul 12.5ul

[0204] (4) After staining, wash with 2% Triton X-100 three times, each time for 10-15 minutes, wash twice with PBS, stain with DAPI (1:1000) for 5-10 minutes, and seal with 50% glycerol. Figure 6 .

[0205] 6. Scratch assay of U251 cells overexpressing circ-PHF21A

[0206] (1) First, use a marker pen and a ruler to evenly draw horizontal lines on the back of the 6-well plate, approximately every 0.5 to 1 cm, across the holes. Make at least 5 lines in each hole.

[0207] (2) Plant approximately 4×10 5 Cells were cultured overnight in a 37°C, 5% CO2 cell incubator.

[0208] (3) After cell transfection, wait until the cells reach the logarithmic growth phase, digest with an appropriate amount of trypsin-EDTA mixture, and adjust to a single cell suspension using a pipette. A certain number of cells are inoculated in 6-well plates according to the grouping.

[0209] (4) When the cells are growing logarithmically, aspirate the culture medium and use a sterile 200 μl pipette tip to scratch the horizontal line on the bottom of the six-well plate. Apply even force to ensure uniform width.

[0210] (5) Wash the cells three times with PBS, remove the scratched cells, add serum-free medium, and culture in a cell culture incubator at 37°C and 5% CO2; take pictures at 0h, 24h, and 48h, measure the scratch width, and record the cell migration. Figure 7 .

[0211] VII. Transwell invasion assay of U87 and U251 cells overexpressing circ-PHF21A

[0212] (1) Cells transfected with the circ-PHF21A overexpressing plasmid were digested with trypsin-EDTA solution, centrifuged at 800 rpm for 3 minutes, resuspended in serum-free DMEM medium, counted, and adjusted to a cell density of 1×10 6 / ml, add 100ul serum-free DMEM medium cell suspension to the upper chamber of the Transwell chamber, and add 600ul complete culture medium to the lower chamber.

[0213] (2) Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0214] (3) Remove the small chamber and wipe off the cells in the upper chamber with a cotton swab.

[0215] (4) Fix with 4% paraformaldehyde for 15 minutes, wash once with PBS, stain with crystal violet for 10 minutes, wash once with PBS, and check whether the cells pass through the small holes. If they do, terminate the other experimental groups and take pictures for statistics. Figure 8 .

[0216] The present invention also relates to a GBM diagnostic kit, which comprises primers capable of amplifying the circular RNA molecule circ-PHF21A. The amplification primers may be a primer pair consisting of the DNA sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 or a primer pair consisting of the DNA sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0217] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

[0218] SEQ ID NO: 1

[0219] GGGGCTAGAGAGCTGAAGGAGAGCCAGTTTCCCCAAAATTGCTGCAGTGAGAAGGAGGGTTTGTTACT

[0220] TTAAACAGAGGCTGAAGAAACTATAGAATTAGCAGAGAAAGTGGAGAAGGTAGAGGATGGAGTTGCAG

[0221] ACTCTACAGGAGGCTCTTAAAGTGGAAATTCAGGTTTCACCAGAAACTGGTTGCTCAAATGAAGCAGGA

[0222] <h2 style=";text-align:left;direction:ltr">TCCACAGAATGCTGACTTAAAGAAACAGCTTCATGAACTCCAAGCCAAAATCACAGCTTTGAGTGAGA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0223] <h2 style=";text-align:left;direction:ltr"> AACAG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0224] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0225] <h2 style=";text-align:left;direction:ltr"> 5'-GGTAGAGGATGGAGTTGCAGA-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0226] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0227] <h2 style=";text-align:left;direction:ltr"> 5'-AGCTGTGATTTTGGCTTGGA-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0228] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0229] <h2 style=";text-align:left;direction:ltr"> 5'-CTCCAAGCCAAAATCACAGC-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0230] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0231] <h2 style=";text-align:left;direction:ltr"> 5'-GGGAAACTGGCTCTCCTTCA-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0232] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0233] <h2 style=";text-align:left;direction:ltr"> GAPDH-F:AGAAGGCTGGGGCTCATTTG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0234] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0235] <h2 style=";text-align:left;direction:ltr"> GAPDH-R:GCAGGAGGCATTGCTGATGAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0236] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0237] <h2 style=";text-align:left;direction:ltr"> hsa_circ_0000296-3xFlag-F2:TCACCAGAAACTGGTTGCTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0238] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0239] <h2 style=";text-align:left;direction:ltr"> hsa_circ_0000296-3xFlag-R2:AGCTCTCTAGCCCCCTGTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0240] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0241] <h2 style=";text-align:left;direction:ltr"> hsa_circ_0000296-3xFlag-F2:TCACCAGAAACTGGTTGCTC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0242] SEQ ID NO:11

[0243] hsa_circ_0000296-3xFlag-R1:ACCTTCTCCACTTTCTCTGC

[0244] SEQ ID NO:12

[0245] >hsa_circ_0000296-3xFlag|355nt(pLC5-ciR)

[0246] GGGGCTAGAGAGCTGAAGGAGAGCCAGTTTCCCCAAAATTGCTGCAGTGAGAAGAGGAGTTTGTTACT

[0247] TggtggttctggtGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATG

[0248] ACGATGACAAGTAAACAGAGGCTGAAGAAACTATAGAATTAGCAGAGAAAGTGGAGAAGGTAGAGGAT

[0249] GGAGTTGCAGACTCTACAGGAGGCTCTTAAAGTGGAAATTCAGGTTCACCAGAAACTGGTTGCTCAAA

[0250] TGAAGCAGGATCCACAGAATGCTGACTTAAAGAAACAGCTTCATGAACTCCAAGCCAAAATCACAGCT

[0251] TTGAGTGAGAAACAG

[0252] SEQ ID NO:13

[0253] >3xFlag

[0254] GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAG

[0255] SEQ ID NO:14

[0256] translation=DYKDHDGDYKDHDIDYKDDDDK

[0257] SEQ ID NO:15

[0258] Linker GGSG:ggtggttctggt

[0259] SEQ ID NO:16

[0260] Translation product: >ORF (100 aa, 11.48 kDa)

[0261] MELQTLQEALKVEIQVHQKLVAQMKQDPQNADLKKQLHELQAKITALSEKQGARELKESQFPQNCCSEKRSLLLGGSGDYKDHDGDYKDHDIDYKDDDDK.

[0262] SEQ ID NO:17

[0263] PHF21A - 74aa

[0264] MELQTLQEALKVEIQVHQKLVAQMKQDPQNADLKKQLHELQAKITALSEKQGARELKESQFPQNCCSE

[0265] KRSLLL。

Claims

1. Use of the circular RNA molecule circ-PHF21A in the preparation of a glioblastoma diagnostic kit, characterized in that: The CircBase ID of the circ-PHF21A is hsa_circ_0000296, the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO: 1, and the structure of the circular RNA molecule circ-PHF21A is a circular structure formed by connecting the nucleotide sequence shown in SEQ ID NO: 1 end to end.

2. Use of the circular RNA molecule circ-PHF21A in the preparation of a drug for treating glioblastoma, characterized in that: The CircBase ID of the circ-PHF21A is hsa_circ_0000296, the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO: 1, and the structure of the circular RNA molecule circ-PHF21A is a circular structure formed by connecting the nucleotide sequence shown in SEQ ID NO: 1 end to end.

3. A glioblastoma diagnostic kit, characterized in that: The kit comprises primers capable of amplifying the circular RNA molecule circ-PHF21A, and the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO:

1.

4. The glioblastoma diagnostic kit according to claim 3, characterized in that: The amplification primers are a primer pair consisting of the DNA sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 or a primer pair consisting of the DNA sequences shown in SEQ ID NO: 4 and SEQ ID NO:

5.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a circular RNA molecule circ-PHF21A, and the nucleotide sequence of the circular RNA molecule circ-PHF21A is shown in SEQ ID NO:

1.

6. A polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

17.

7. Use of the polypeptide according to claim 6 in preparing a glioblastoma diagnostic kit.

8. Use of the polypeptide according to claim 6 in the preparation of a medicament for treating glioblastoma.

Images