Glioma biomarker and use thereof

Abstract

The application provides a Circ-SLC25A24 which is highly expressed in glioma and has coding function and a Circ-SLC25A24-19KD coded by the Circ-SLC25A24. By inhibiting the Circ-SLC25A24 nucleotide through siRNA, shRNA or AS O, the proliferation, migration, invasion and the like of glioma cells can be significantly inhibited. The Circ-SLC25A24 or the Circ-SLC25A24-19KD can be used as a glioma diagnosis marker and a treatment target. The Circ-SLC25A24-19KD amino acid sequence coded by the ORF of the Circ-SLC25A24 across a splicing site has specificity and can be used as a specific target of a molecular targeted drug, provides a new idea for the research and development of a drug for treating glioma, and has important clinical medication value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tumor markers, and particularly relates to a glioma biomarker and application thereof. BACKGROUND

[0002] Glioma is the most common primary central nervous system malignancy, accounting for about 80% of all central nervous system malignancies. At present, the treatment of glioma is mainly surgery combined with radiotherapy, chemotherapy and other comprehensive treatment methods, but the prognosis of patients is still poor, and the median survival is only 12.1-14.6 months, and only 3-5% of patients can survive for more than 3 years. Since the current treatment methods still cannot significantly improve the survival of patients, discovering and elucidating the mechanism of occurrence and development of glioma has become the focus of the current glioma research field. We expect to provide new ideas for clinical treatment and lay the foundation for developing effective treatment programs by revealing the occurrence and development process of glioma.

[0003] In recent years, with the development of high-throughput sequencing technology and bioinformatics technology, a large number of circular RNAs (circRNAs) have been found in the human body. Circular RNA is formed by reverse splicing of precursor RNA and has a closed loop structure, and has important biological functions. Because there is no exposed end structure, it is resistant to the cutting of exonuclease RNase R, and compared with linear RNA molecules, circular RNA can exist more stably in the organism and play its biological functions. Circular RNA has the following biological functions, such as regulating gene transcription and splicing in the nucleus; as a "sponge" to adsorb miRNA and inhibit its function; circRNA can bind to proteins to regulate the activity and function of proteins. In addition, circular RNA can also encode proteins as a translation template through its own ribosome intervention site, and the proteins encoded by circular RNA also have important biological functions. SUMMARY

[0004] Therefore, the present application aims to overcome the defects in the prior art and provide a biomarker for diagnosis or prognosis of brain glioma and application thereof.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] The first object of the present application is to provide a glioma biomarker, which is circular RNA and / or small molecule protein, the circular RNA is named Circ-SLC25A24, and its base sequence is shown as SEQ NO: 1; the small molecule protein is named Circ-SLC25A24-19KD, and the small molecule protein is encoded by the circular RNA, and its amino acid sequence is shown as SEQ NO: 2.

[0007] A second object of the present application is to provide a specific primer pair for amplifying Circ-SLC25A24, the nucleotide sequence of which is shown as SEQ ID NO. 3 and SEQ ID NO. 4, or as SEQ ID NO. 5 and SEQ ID NO. 6, or as SEQ ID NO. 7 and SEQ ID NO. 8.

[0008] A third object of the present application is to provide use of the above-mentioned tumor biomarker in the preparation of a product for diagnosing and / or screening and / or predicting and / or prognosing glioma.

[0009] Preferably, the product comprises a detection reagent or kit for diagnosing and / or screening and / or predicting and / or prognosing evaluation of glioma, both of which comprise the above-mentioned specific primer pair for amplifying Circ-SLC25A24.

[0010] Preferably, the detection reagent and kit further comprise a reference primer, which is a primer taking GAPDH as a reference, and the nucleotide sequence of the reference primer is shown as SEQ ID NO. 9 and SEQ ID NO. 10.

[0011] A fourth object of the present application is to provide a Circ-SLC25A24 expression inhibitor, which is an siRNA, shRNA or ASO for inhibiting the expression of the above-mentioned Circ-SLC25A24.

[0012] Preferably, the nucleotide sequence of the siRNA is shown as SEQ ID NO. 11 or SEQ ID NO. 12.

[0013] Preferably, the nucleotide sequence of the shRNA is shown as SEQ ID NO. 13 or SEQ ID NO. 14.

[0014] Preferably, the nucleotide sequence of the ASO is shown as SEQ ID NO. 15 or SEQ ID NO. 16.

[0015] A fifth object of the present application is to provide use of the above-mentioned Circ-SLC25A24 expression inhibitor in the preparation of a drug for preventing and / or treating glioma.

[0016] SLC25A24 can encode a mitochondrial inner membrane ATP-Mg / Pi carrier, also known as short Ca 2+SCaMC1. SCaMC-1 is the major isoform of ATP-Mg / Pi carrier in cancer cells and is highly overexpressed in a range of in vivo tumors and cell lines. Mitochondrial permeability transition (mPT) plays a central role in permeabilizing the inner mitochondrial membrane (IMM) and leading to cell necrosis. Through SCaMC-1, cytosolic Ca 2+ ([Ca 2+ ]cyt) mediated ATP / ADP uptake increases mitochondrial Ca 2+ buffering, thus contributing to tumor cell resistance to mPT. Downregulation of SLC25A24 leads to a dramatic decrease in mitochondrial Ca 2+ buffering capacity and sensitizes cells to mPT-mediated necrotic death induced by oxidative stress and Ca 2+ overload, promoting cancer cell death.

[0017] At present, the related art does not disclose the expression of the circular RNA subtype Circ-SLC25A24 of SLC25A24 and the small molecule protein Circ-SLC25A24-19KD encoded by the same in specific tumors, nor does it disclose whether Circ-SLC25A24 and the small molecule protein Circ-SLC25A24-19KD can be used for specific tumor diagnosis and prognosis related research.

[0018] Compared with the prior art, the present application has the following advantages:

[0019] The Circ-SLC25A24 and Circ-SLC25A24-19KD of the present application can be used as markers for diagnosis / screening / prediction / prognosis of glioma, and can also be used as new therapeutic targets for glioma, thereby providing new treatment ideas and schemes for glioma patients. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A is the structure map of Circ-SLC25A24 of the present application; Figure 1 B is the nucleotide sequence and circular structure Sanger sequencing identification result of Circ-SLC25A24; Figure 1 C is the formation mode diagram of SLC25A24 circular RNA; Figure 1 D is the nucleotide sequence and circular structure Sanger sequencing identification result of Circ-SLC25A24;

[0021] Figure 2 A is the expression of Circ-SLC25A24 in glioma cell lines; Figure 2 B is the expression difference of Circ-SLC25A24 in low-grade and high-grade glioma;

[0022] Figure 3 A is the Circ-SLC25A24 translation small molecule protein pattern diagram and the sequence of the small molecule protein; Figure 3 B is the IRES activity detection of Circ-SLC25A24; Figure 3 C is the detection of Circ-SLC25A24-Flag;

[0023] Figure 4 A is the siRNA knockdown rate detection result of Circ-SLC25A24, Figure 4 B is the antisense nucleotide ASO knockdown rate detection result, Figure 4 C is the shRNA knockdown rate detection result. ***: P<0.001, ****: P<0.0001;

[0024] Figure 5 is the CCK-8 cell proliferation experiment result, ****: P<0.0001;

[0025] Figure 6 is the Edu detection result;

[0026] Figure 7 is the cell invasion analysis experiment result;

[0027] Figure 8 is the cell scratch experiment result;

[0028] Figure 9 is the animal tumorigenesis experiment result, wherein, Figure 9 A is the tumor volume difference of tumors of rats in each group, Figure 9 B is the tumor weight difference of tumors of rats in each group. DETAILED DESCRIPTION

[0029] Unless defined, the technical terms used in the following examples have the same meanings as generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods used, unless otherwise specified, are all conventional methods.

[0030] The present invention will be described in detail below in conjunction with examples.

[0031] Example 1 Differentially expressed Circ-SLC25A24 and verification thereof

[0032] The present invention uses chip sequencing (Arraystar Human circRNA Array V2, Kangcheng Bio, Shanghai) technology to analyze the differential expression of circular RNA in four pairs of glioma tissues and normal brain tissues. The sequencing results are as follows: Figure 1As shown in A, 1B, it can be seen that Circ-SLC25A24 is significantly highly expressed in glioma samples compared with normal tissues.

[0033] As shown in Figure 1 As shown in C, 1D, analysis by UCSC (http: / / genome.ucsc.edu / ) online database software found that Circ-SLC25A24 gene is located in the short arm of human chromosome 1, chr1 (p13.3) region, with a genome spanning 13015 bp, derived from exons 4-7 of human SLC25A24 gene; according to the information of circular RNA generated by SLC25A24 collected by the circular RNA authority database circBase (http: / / circrna.org / ), the circRNA ID of Circ-SLC25A24 is hsa_circ_0004270, the mature circular RNA sequence is 532 nt, and it is named Circ-SLC25A24, and its sequence is as follows: CATTGATGTTGATGGGACAATGACAGTGGACTGGAATGAATGGAGAGACTACTTCTTATTTAATCCTGTTACAGACATTGAGGAAATTATCCGTTTCTGGAAACATTCTACAGGAATTGACATAGGGGATAGCTTAACTATTCCAGATGAATTCACGGAAGACGAAAAAAAATCCGGACAATGGTGGAGGCAGCTTTTGGCAGGAGGCATTGCTGGTGCTGTCTCTCGAACAAGCACTGCCCCTTTGGACCGTCTGAAAATCATGATGCAGGTTCACGGTTCAAAATCAGACAAAATGAACATATTTGGTGGCTTTCGACAGATGGTAAAAGAAGGAGGTATCCGCTCGCTTTGGAGGGGAAATGGTACAAACGTCATCAAAATTGCTCCTGAGACAGCTGTTAAATTCTGGGCATATGAACAGTACAAGAAGTTACTTACTGAAGAAGGACAAAAAATAGGAACATTTGAGAGATTTATTTCTGGTTCCATGGCTGGAGCAACTGCACAGACTTTTATATATCCAATGGAG;

[0034] The accurate circularization site of Circ-SLC25A24 was obtained by designing three pairs of PCR amplification primers on both sides of the circular RNA reverse connection site, amplifying the sequences on both sides of the circularization site of the circular RNA, and using the sanger DNA sequencing method. The specific PCR amplification primer sequences for amplifying Circ-SLC25A24 are as follows:

[0035] 1. Circ-SLC25A24-F: 5' TGGAGCAACTGCACAGACTT 3',

[0036] Circ-SLC25A24-R: 5' CCAGAAACGGATAATTTCCTCA 3';

[0037] 2. Circ-SLC25A24-F: 5' CTGGAGCAACTGCACAGACTT 3',

[0038] Circ-SLC25A24-R: 5' CCAGAAACGGATAATTTCCTCA 3';

[0039] 3. Circ-SLC25A24-F: 5' GGAGCAACTGCACAGACTTTT 3',

[0040] Circ-SLC25A24-R: 5' CCAGAAACGGATAATTTCCTCA 3'.

[0041] The amplification product size of the primer is 137, 138, 136 bp; GAPDH is selected as the internal reference correction gene, and the primer sequences are as follows:

[0042] GAPDH-F: 5' GGTGGTCTCCTCTGACTTCAACA 3'

[0043] GAPDH-R: 5' GTTGCTGTAGCCAAATTCGTTGT 3'

[0044] The amplification product size of the primer is 127 bp;

[0045] The cDNA of brain glioma cell line U87 was used as a template for PCR amplification. The reaction system and conditions for PCR amplification of the target fragment are described as follows: the total system is 20 microliters, specifically 10 microliters of 2x PCR MIX (biomark company), 1 microliter of each of the upstream and downstream primers (10 mM), 2 microliters of cDNA template, and the rest is made up with sterilized water to 20 microliters of system. The reaction conditions are as follows: 95°C for 3 min for pre-denaturation, 95°C for 15 s for denaturation, 60°C for 40 s for annealing, 95°C for 15 s for extension, a total of 40 cycles, and then 72°C for 5 min for continued extension after the PCR reaction cycle, and then 16°C for storage. The PCR product is purified and subjected to sanger DNA sequence determination. The accurate splicing points of the circular RNA are identified by the sanger DNA sequencing method.

[0046] Example 2 Expression of Circ-SLC25A24

[0047] RT-QPCR was used to detect the expression of Circ-SLC25A24 in glioma cell lines (U87, LN229, U251, A172, SNB19, LN18) and human normal astrocytes, and the results are shown in Figure 2 A, it can be seen that Circ-SLC25A24 is highly expressed in glioma cell lines (U87, LN229, U251, LN18). RT-QPCR was used to detect the expression of Circ-SLC25A24 in different grades of glioma. Among them, the WHO grade Ⅰ-Ⅱ of glioma is low-grade glioma (LGG), and Ⅲ-Ⅳ is high-grade glioma (HGG), and the results are shown in Figure 2 B, it can be seen that the expression of Circ-SLC25A24 in high-grade glioma is higher.

[0048] Example 3 Prediction and verification of Circ-SLC25A24 translated protein

[0049] Circ-SLC25A24 may have the potential to encode proteins, and the prediction found that Circ-SLC25A24 mature RNA sequence 532 nt, containing a ribosome entry site (IRES) and open reading frame (ORF), can theoretically translate into a small molecule protein composed of 172 amino acids. The C-terminal of the protein translated by Circ-SLC25A24 has an extra tail of 1 amino acid (H: histidine His), which is a unique terminal amino acid sequence of Circ-SLC25A24 compared to the protein translated by SLC25A24. The molecular weight of the new protein translated by Circ-SLC25A24 was predicted to be about 19KD by protein molecular weight prediction software (http: / / www.bio-soft.net / sms / prot_mw.html), and it was named Circ-SLC25A24-19KD, as shown in Figure 1B. Figure 3 A, the sequence of which is shown as follows: MTVDWNEWRDYFLFNPVTDIEEIIRFWKHSTGIDIGDSLTIPDEFTEDEKKSGQWWRQLLAGGIAGAVSRTSTAPLDRLKIMMQVHGSKSDKMNIFGGFRQMVKEGGIRSLWRGNGTNVIKIAPETAVKFWAYEQYKKLLTEEGQKIGTFERFISGSMAGATAQTFIYPMEH.

[0050] The IRES sequence of Circ-SLC25A24 was cloned between Rluc and Luc reporter gene to construct a dual luciferase reporter plasmid, and a blank plasmid was used as a control. 5000 glioma cells U251 were seeded in a 24-well plate culture plate, and the cells were transfected 24 h after adhesion. Before transfection, 25 microliters of serum-free medium DMEM, 0.3 micrograms of plasmid and 0.4 microliters of P3000 were prepared into a mixed solution; 25 microliters of serum-free medium DMEM and 0.8 microliters of lipo3000 liposome were uniformly mixed to make a liposome mixed solution; the above two mixed solutions were mixed in equal proportions, and were placed at room temperature for 10 min; the operation was performed according to the operation instruction of the transfection reagent (LipofectamineTM3000 Transfection Reagent, ThermoFisher Scientific, #2367427); the final liquid volume in the 24-well plate was 500 microliters, and the cells were transfected for 8 hours, and then 500 microliters of normal culture medium (10% fetal bovine serum plus 90% DMEM medium) was added, and the cells were cultured at 37 degrees and 5% carbon dioxide. The results showed that Figure 3 B. Compared with the blank plasmid, the IRES of Circ-SLC25A24 induced higher Luc / Rluc activity.

[0051] A Circ-SLC25A24-Flag plasmid with a Flag tag sequence was constructed and transfected into 293T cells and U87 cells. The total protein of the cells was extracted with RIPA lysis buffer, and the extracted protein was quantified by BCA protein quantification method; 5% SDS-PAGE concentrated gel and 15% SDS-PAGE separation gel were configured, and 30 micrograms of total protein was loaded; the protein electrophoresis was run at 80V for 30 min and 120V for 1 h; the membrane transfer was performed at 360A for 1 h; 5% skim milk was blocked at room temperature for 2 h; Flag rabbit monoclonal antibody (Affinity Biosciences Cat#T0053, RRID:AB_2843447) (1:1000), GAPDH antibody (Affinity Biosciences Cat#AF7021, RRID:AB_2839421) (1:3000); 4 degrees overnight incubation; the next day, the secondary antibody (1:10000) was incubated at room temperature for 1 h, washed with TBST for 5 times for 5 min each time, and then light emission, development and fixation were performed. Western blotting detected the expression of Flag, see Figure 3 C. As shown above, Circ-SLC25A24 has the potential to encode proteins using its IRES and ORF.

[0052] Example 4 Cell culture and transfection

[0053] 1. Design and preparation of siRNA, shRNA and antisense nucleotide ASO

[0054] Shanghai Jima Pharmaceutical Technology Co., Ltd. is entrusted to design and chemically synthesize siRNA, shRNA and antisense nucleotide ASO according to the splicing site of Circ-SLC25A24, and to make chemical modification of 2-oxymethyl and phosphorothioate of nucleotide, enhance the ability of anti-nuclease activity, and improve the stability of small nucleic acid.

[0055] The sequences of siRNA, shRNA and antisense nucleotide ASO are as follows:

[0056] The sequence of siRNA is as follows:

[0057] siRNA-1 CAAUGGAGCAUUGAUGUUGTT;

[0058] siRNA-2 AUCCAAUGGAGCAUUGAUGTT;

[0059] The sequence of shRNA is as follows:

[0060] shRNA-1 CAATGGAGCATTGATGTTG;

[0061] shRNA-2 ATCCAATGGAGCATTGATG;

[0062] The sequence of ASO is as follows:

[0063] ASO-1 CAUCAATGCTCCATTGGAUA;

[0064] ASO-2 CAACATCAATGCTCCAUUGG;

[0065] 2. Cell culture and transfection

[0066] U87 glioma cells were seeded in 6-well plates at a density of 300,000 cells per well. After the cells adhered to the well, transfection was performed. Before transfection, 100 microliters of serum-free medium DMEM and siRNA or ASO were mixed to form a mixed solution. 100 microliters of serum-free medium DMEM and 5 microliters of RNAiMAX liposome were uniformly mixed to form a liposome mixed solution. The two mixed solutions were mixed in equal proportions and placed at room temperature for 10 min. The transfection reagent (Lipofectamine RNAiMAX, ThermoFisher Scientific, #13778150) was used according to the manufacturer's instructions. The final liquid volume in the 6-well plate was 2 ml, the final concentration of siRNA or ASO was 100 nM, and the transfection was performed for 8 hours. Then, 1 ml of normal culture medium (10% fetal bovine serum plus 90% DMEM medium) was added, and the cells were cultured at 37 degrees Celsius in 5% carbon dioxide.

[0067] U87 glioma cells were seeded in 24-well plates at a density of 20,000 cells per well. After the cells adhered to the well, transfection was performed. 1 ug (50 pmol) of shRNA was added to a certain amount of serum-free DMEM diluent, mixed thoroughly, and an RNA diluent was prepared with a final volume of 25 microliters. 1.5 microliters of EntransterTM-R4000 (Engreen Biosystem, Beijing, China) was then added to 24 microliters of serum-free DMEM diluent, mixed thoroughly, and an EntransterTM-R4000 diluent was prepared with a final volume of 25 microliters. The mixture was incubated at room temperature for 5 minutes. The EntransterTM-R4000 diluent and the shRNA diluent were mixed thoroughly and incubated at room temperature for 15 minutes. The transfection complex was prepared. 50 microliters of the transfection complex was added to the cells in 0.45 ml of normal culture medium and mixed evenly. After transfection, the cell state was observed 6 hours later, the culture medium was replaced, and the cells were cultured for another 48 hours. The cells were cultured at 37 degrees Celsius in 5% carbon dioxide.

[0068] After transfection of siRNA, antisense nucleotide ASO, and shRNA, respectively, the results are shown in Figure 4 As shown in A-4C, the content of Circ-SLC25A24 was significantly reduced.

[0069] Example 5 Cell Proliferation Experiment

[0070] Circ-SLC25A24 overexpression plasmid, IRES deleted Circ-SLC25A24 Del IRES plasmid and SLC225A24 172aa plasmid expressing Circ-SLC25A24-19KD were constructed, and U87 cells were transfected with blank plasmid as control. Cells in different groups were plated in 96-well plates, 2000 cells per well, 5 replicates per group. After the cells adhered and grew, the medium containing 10% CCK-8 was replaced at the same time every day, and the absorbance at 450 nm was detected after 2 hours. Finally, the absorbance at different time points was standardized according to the absorbance on the first day. Cell activity was detected at different time points (CCK-8 experiment), and the results are shown in Figure 5 As can be seen, compared with the control group and the Circ-SLC25A24 Del IRES group, the proliferation ability of U87 cells transfected with Circ-SLC25A24 overexpression plasmid and SLC225A24 172aa plasmid was significantly up-regulated, indicating that Circ-SLC25A24-19KD but not Circ-SLC25A24 could promote the proliferation of glioma cells.

[0071] Example 6 EdU uptake experiment

[0072] Different groups of cells were plated in 24-well plates, 20,000 cells per well, 3 replicates per group. After the cells adhered, U87 cells were transfected with ASO, the final concentration of ASO was 100 nM, and the transfection time was 8 hours. Then 1 ml of normal culture medium (10% fetal bovine serum plus 90% DMEM medium) was added and cultured for 24 hours. The cell culture conditions were 37 degrees and 5% carbon dioxide. According to the operation instruction of BeyoClickTM EdU-594 cell proliferation detection kit (Beyotime), EdU was incubated and fluorescently labeled, and finally the uptake of EdU was detected under a fluorescence microscope. After knocking down Circ-SLC25A24, the EdU uptake was detected. The results are shown in Figure 6 As can be seen, after knocking down Circ-SLC25A24, the EdU uptake of the cells decreased, indicating that the proliferation ability decreased significantly.

[0073] Example 7 Cell invasion experiment

[0074] U87 glioma cells were seeded in 6-well plates at a density of 300,000 cells per well. After the cells adhered, siRNA was transfected at a final concentration of 100 nM. The cells were cultured for 8 hours, then 1 ml of normal culture medium (10% fetal bovine serum plus 90% DMEM medium) was added, and the cells were cultured at 37°C in 5% CO2 for 24 hours. 30 μg of Martrigel was added to the upper chamber of a Transwell chamber, and 20,000 siRNA-transfected U87 glioma cells were added. The cells were cultured at 37°C in 5% CO2 for 12 hours, then the filter membrane was fixed with ethanol, stained with PE, and photographed to count the number of cells that had passed through the Martrigel. The results are shown in Figure 7 It can be seen that the migration ability of U87 cells after the addition of siRNA was significantly weakened, and siRNA had a significant inhibitory effect on the migration ability of U87 cells.

[0075] Example 8 Cell Scratch Test

[0076] U87 glioma cells were seeded in 6-well plates at a density of 300,000 cells per well. After the cells adhered, siRNA was transfected at a final concentration of 100 nM. The cells were cultured for 8 hours, then 1 ml of normal culture medium (10% fetal bovine serum plus 90% DMEM medium) was added, and the cells were cultured at 37°C in 5% CO2 for 24 hours. 30 μg of Martrigel was added to the upper chamber of a Transwell chamber, and 20,000 siRNA-transfected U87 glioma cells were added. The cells were cultured at 37°C in 5% CO2 for 12 hours, then the filter membrane was fixed with ethanol, stained with PE, and photographed to count the number of cells that had passed through the Martrigel. The results are shown in Figure 8 It can be seen that the migration ability of U87 cells after the addition of siRNA was significantly weakened, and siRNA had a significant inhibitory effect on the migration ability of U87 cells.

[0077] Example 9 Animal Tumor Formation Test

[0078] shRNA was delivered into glioma cells U87 cells using a lentivirus infection method to stably express, a stable transfection cell line was constructed, and Circ-SLC25A24 was continuously knocked down. Glioma cells U87 cells transfected with shRNA were injected subcutaneously into 4-week-old BALB / c female nude mice (Beijing Hengfu Biotechnology Co., Ltd.) at a cell amount of 4 million, to construct a glioma xenograft animal model. The 9 nude mice were divided into 3 groups, 3 mice in each group, namely the NC group, the shRNA#1 group, and the shRNA#2 group. Then, the tumor volume was measured every 2 days. The nude mice were sacrificed after 21 days, the subcutaneous tumor tissue was taken, the size and weight of the tumor were measured, and the tumor tissue was fixed with paraformaldehyde, paraffin-embedded, and HE-stained sections were prepared. The results are shown in Figure 9 It can be seen that the tumor volume and weight of the shRNA-transfected group were significantly smaller than those of the NC group.

[0079] The above merely provides the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> General Hospital of Tianjin Medical University <120> A glioma biomarker and application thereof <141> 2022-06-08 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 532 <212> DNA <213> Artificial Sequence <400> 1 cattgatgtt gatgggacaa tgacagtgga ctggaatgaa tggagagact acttcttatt 60 taatcctgtt acagacattg aggaaattat ccgtttctgg aaacattcta caggaattga 120 cataggggat agcttaacta ttccagatga attcacggaa gacgaaaaaa aatccggaca 180 atggtggagg cagcttttgg caggaggcat tgctggtgct gtctctcgaa caagcactgc 240 ccctttggac cgtctgaaaa tcatgatgca ggttcacggt tcaaaatcag acaaaatgaa 300 catatttggt ggctttcgac agatggtaaa agaaggaggt atccgctcgc tttggagggg 360 aaatggtaca aacgtcatca aaattgctcc tgagacagct gttaaattct gggcatatga 420 acagtacaag aagttactta ctgaagaagg acaaaaaata ggaacatttg agagatttat 480 ttctggttcc atggctggag caactgcaca gacttttata tatccaatgg ag 532 <210> 2 <211> 172 <212> PRT <213> Artificial Sequence <400> 2 Met Thr Val Asp Trp Asn Glu Trp Arg Asp Tyr Phe Leu Phe Asn Pro 1 5 10 15 Val Thr Asp Ile Glu Glu Ile Ile Arg Phe Trp Lys His Ser Thr Gly 20 25 30 Ile Asp Ile Gly Asp Ser Leu Thr Ile Pro Asp Glu Phe Thr Glu Asp 35 40 45 Glu Lys Lys Ser Gly Gln Trp Trp Arg Gln Leu Leu Ala Gly Gly Ile 50 55 60 Ala Gly Ala Val Ser Arg Thr Ser Thr Ala Pro Leu Asp Arg Leu Lys 65 70 75 80 Ile Met Met Gln Val His Gly Ser Lys Ser Asp Lys Met Asn Ile Phe 85 90 95 Gly Gly Phe Arg Gln Met Val Lys Glu Gly Gly Ile Arg Ser Leu Trp 100 105 110 Arg Gly Asn Gly Thr Asn Val lie Lys lie Ala Pro Glu Thr Ala Val 115 120 125 Lys Phe Trp Ala Tyr Glu Gin Tyr Lys Lys Leu Leu Thr Glu Glu Gly 130 135 140 Gln Lys lie Gly Thr Phe Glu Arg Phe lie Ser Gly Ser Met Ala Gly 145 150 155 160 Ala Thr Ala Gin Thr Phe lie Tyr Pro Met Glu His 165 170 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tggagcaact gcacagactt 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 ccagaaacgg ataatttcct ca 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 ctggagcaac tgcacagact t 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 ccagaaacgg ataatttcct ca 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 ggagcaactg cacagacttt t 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <400> 8 ggagcaactg cacagacttt t 21 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 ggtggtctcc tctgacttca aca 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <400> 10 gttgctgtag ccaaattcgt tgt 23 <210> 11 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 11 caauggagca uugauguugt t 21 <210> 12 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 12 auccaaugga gcauugaugt t 21 <210> 13 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 13 caatggagca ttgatgttg 19 <210> 14 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 14 atccaatgga gcattgatg 19 <210> 15 <211> 20 <212> DNA / RNA <213> Artificial Sequence <400> 15 caucaatgct ccattggaua 20 <210> 16 <211> 20 <212> DNA / RNA <213> Artificial Sequence <400> 16 caacatcaat gctccauugg 20

Claims

1. The use of a reagent for detecting glioma biomarkers in tissues in the preparation of products for the diagnosis, screening, or prediction of gliomas, characterized in that: The biomarker is a circular RNA and / or a small protein. The circular RNA is named Circ-SLC25A24, and its nucleotide sequence is shown in SEQ NO:

1. The small protein is named Circ-SLC25A24-19KD, and the small protein is encoded by the circular RNA, and its amino acid sequence is shown in SEQ NO:

2.

2. The application according to claim 1, characterized in that: The product is a specific primer set for amplifying Circ-SLC25A24, and the nucleotide sequences of the specific primer set are shown in SEQ ID NO.3 and SEQ ID NO.4, or in SEQ ID NO.5 and SEQ ID NO.6, or in SEQ ID NO.7 and SEQ ID NO.

8.

3. The application according to claim 1, characterized in that: The product includes diagnostic reagents or kits for diagnosing and / or screening and / or predicting gliomas, wherein the diagnostic reagents and kits each include the specific primer set for amplifying Circ-SLC25A24 as described in claim 2; the diagnostic reagents and kits also include internal control primers, wherein the internal control primers are primers with GAPDH as an internal control, and the nucleotide sequences of the internal control primers are shown in SEQ ID NO. 9 and SEQ ID NO.

10.

4. The use of a Circ-SLC25A24 expression inhibitor in the preparation of drugs for the prevention and / or treatment of gliomas, characterized in that: The expression inhibitor is a siRNA, shRNA, or ASO used to inhibit the expression of Circ-SLC25A24 according to claim 1; the nucleotide sequence of the siRNA is shown in SEQ ID NO.11 or SEQ ID NO.12; the nucleotide sequence of the shRNA is shown in SEQ ID NO.13 or SEQ ID NO.14; and the nucleotide sequence of the ASO is shown in SEQ ID NO.15 or SEQ ID NO.16.

Images