IncATRIG inhibitor and application thereof in preparation of medicine for treating glioma

Abstract

The invention discloses an lncATRIG inhibitor and an application of the lncATRIG inhibitor in preparation of a medicine for treating glioma. The lncATRIG inhibitor comprises one or more of the following sequences: LNA-1, LNA-2 and LNA-3 as shown in the specification; wherein the sequence of the LNA-1 is as shown in Seq ID (Identity) No. 2; the sequence of the LNA-2 is as shown in Seq ID No. 3; the sequence of the LNA-3 is as shown in Seq ID (Identity) No.4; the cDNA (complementary deoxyribonucleic acid) sequence of the lncATRIG is as shown in Seq ID (identity) No. 1. The lncATRIG inhibitor provided by the invention can be used for inhibiting the proliferation of brain glioma cells and promoting the sensitization of temozolomide.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor molecular biology and relates to a long-chain noncoding inhibitor and an application thereof, and specifically relates to a lncATRIG inhibitor and an application thereof in the preparation of a drug for treating glioma and a chemotherapeutic drug sensitizer. Background Art

[0002] Human glioma is the most common primary malignant tumor in the central nervous system, accounting for 80% of all malignant primary brain and central nervous system tumors. Based on histological morphology and molecular characteristics, the World Health Organization (WHO) divides gliomas into types I to IV, of which types I and II are called low-grade gliomas (LGG), while types III to IV include glioblastoma (GBM), anaplastic astrocytoma, and anaplastic oligodendroglioma. Because gliomas have extremely high tolerance to drugs, high recurrence rates, and seriously affect the quality of life of patients, these factors greatly shorten the survival time of patients, so types III and IV gliomas often become the focus of research. The incidence of low-grade gliomas is low, accounting for only 15% of the total number of gliomas. It is well treated by surgical resection, radiotherapy, and chemotherapy, and has a good prognosis. About 47% of patients can survive for more than ten years. In contrast, glioblastoma is one of the most malignant brain tumors. Although its overall incidence is only 5.55 / 100,000, the survival rate of patients is only one-third. The average survival time of patients who do not receive intervention is three months, while the survival time after treatment is 12 to 15 months, and the longest does not exceed five years. Malignant tumors have become the main cause of premature death and shortened life expectancy in many countries.

[0003] Current tumor treatment strategies include surgery, chemotherapy, radiotherapy, and immunotherapy, but there are still many challenges in the treatment process, such as chemotherapy resistance, low drug response rate, and toxic side effects. Temozolomide (TMZ), as a first-line chemotherapy drug, can damage tumor cells by alkylating DNA and is one of the important means of treating GBM. However, long-term use of temozolomide can easily lead to drug resistance, resulting in limited treatment options or even no drugs available after recurrence. The complexity of temozolomide resistance poses a great challenge to the treatment of gliomas, and there is an urgent need to find new molecular targets and treatment strategies.

[0004] During the treatment of temozolomide, drug resistance is the main factor limiting its efficacy. Glioma cells develop resistance to temozolomide through a variety of mechanisms, the most common of which is the enhancement of DNA repair mechanisms. MGMT (O6-methylguanine-DNA methyltransferase) is a key enzyme involved in DNA repair, which can reverse the DNA alkylation damage caused by temozolomide, thereby offsetting the anti-tumor effect of temozolomide. Glioma cells with high expression of MGMT have low sensitivity to temozolomide, indicating a poor prognosis for patients. In addition, temozolomide resistance also involves the regulation of multiple aspects such as the DNA mismatch repair system, apoptosis signaling pathways, and tumor stem cell characteristics. The complex mechanism of drug resistance has prompted researchers to look for new resistance-related factors in order to develop more effective treatment options. Summary of the invention

[0005] In order to solve the above problems, the object of the present invention is to provide a lncATRIG inhibitor.

[0006] Another object of the present invention is to provide applications of lncATRIG inhibitors.

[0007] In order to achieve the above object, the present invention provides a lncATRIG inhibitor, comprising one or more of the sequences LNA-1, LNA-2, and LNA-3 shown below; wherein the sequence of LNA-1 is shown in Seq ID No.2; the sequence of LNA-2 is shown in Seq ID No.3; the sequence of LNA-3 is shown in Seq ID No.4; and the cDNA sequence of the lncATRIG is shown in Seq ID No.1.

[0008] As described above, locked nucleic acid and methoxy modification were performed on each nucleotide of the LNA-1, the LNA-2 and the LNA-3.

[0009] The present invention also provides use of the above-mentioned lncATRIG sequence-targeted inhibitor in preparing a drug for inhibiting the proliferation of brain glioma cells.

[0010] The present invention also provides use of the above-mentioned lncATRIG sequence-targeted inhibitor in the preparation of a temozolomide sensitizing drug.

[0011] The beneficial effects of the present invention are:

[0012] The present invention provides a lncATRIG inhibitor and application thereof. The lncATRIG inhibitor can significantly inhibit the proliferation of drug-resistant glioma cells and enhance their sensitivity to temozolomide. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1AAgarose gel electrophoresis of PCR products from 5'RACE (left) and 3'RACE (right) experiments of lncATRIG.

[0014] Figure 1B Visualization of the gene region of lncATRIG on chromosome VI.

[0015] Figure 1C The expression abundance of the full-length transcript of lncATRIG RNA in U87 and T3-U87 cells detected by Northern blot.

[0016] Figure 1D This is an image of the localization of lncATRIG RNA in human glioma cell lines analyzed by single-molecule fluorescence in situ hybridization.

[0017] Figure 2A The copy number analysis diagram of LncATRIG in different tumor cells.

[0018] Figure 2B The expression of LncATRIG in human glioma tissues, 15 cases of low-grade (LGG) and recurrent (GBM) gliomas.

[0019] Figure 2C Expression of CCAT1 in human glioma tissues, 15 cases of low-grade (LGG) and recurrent (GBM) gliomas.

[0020] Figure 2D To analyze the expression of LncATRIG in human glioma tissue microarray using RNA FISH.

[0021] Figure 2E The figure is a statistical diagram of tissue chip positive cells in normal brain tissue and GBM tissue.

[0022] Figure 3A Comparative photographs showing that overexpression of lncATRIG promoted the colony-forming ability of U87 cells in the presence of TMZ.

[0023] Figure 3B This is a statistical analysis result of the colony-forming ability after lncATRIG overexpression.

[0024] Figure 3C Comparative photographs showing that overexpression of lncATRIG promoted the colony-forming ability of LN229 cells in the presence of TMZ.

[0025] Figure 3D for Figure 3C Statistical analysis results of colony formation ability after lncATRIG overexpression.

[0026] Figure 3E Comparative photographs showing that overexpression of lncATRIG promoted the colony-forming ability of U251 cells in the presence of TMZ.

[0027] Figure 3F for Figure 3E Statistical analysis results of colony formation ability after lncATRIG overexpression.

[0028] Figure 3G This is the result of the cell invasion experiment. Overexpression of lncATRIG in U87 cells enhanced the invasion ability of the cells.

[0029] Figure 3H for Figure 3G Figure 3. Statistical analysis results of cell invasion ability.

[0030] Fig. 3I This is the result of the cell invasion experiment. Overexpression of lncATRIG in LN229 cells enhanced the invasion ability of the cells.

[0031] Figure 3J for Fig. 3I Figure 3. Statistical analysis results of cell invasion ability.

[0032] Figure 3K This is the result of the cell invasion experiment. Overexpression of lncATRIG in U251 cells enhanced the invasion ability of the cells.

[0033] Figure 3L Pictured Figure 3K Figure 3. Statistical analysis results of cell invasion ability.

[0034] Figure 4A After transfecting TMZ-resistant U87 cells with lncATRIG inhibitor, the knockdown efficiency was detected by RT-qPCR.

[0035] Figure 4B Comparative photos of the colony formation experiment results of T3-U87 cells under TMZ treatment conditions after knocking down lncATRIG.

[0036] Figure 4C for Figure 4B Quantitative analysis of the number of colonies.

[0037] Figure 4D Comparative photos of the Transwell assay results of T3-U87 cells after knocking down lncATRIG.

[0038] Figure 4E for Figure 4D Quantitative analysis of the invasion index.

[0039] Figure 5AThis is a statistical graph of the cell resistance to TMZ (IC50) detected by CCK8 assay after knocking down lncATRIG in T3-U87 cells.

[0040] Figure 5B Immunofluorescence images of γ-H2AX and RAD51 in TMZ-treated T3-U87 cells after knockdown of lncATRIG.

[0041] Figure 5C Statistical graph for quantitative analysis of γ-H2AX and RAD51 double-positive Foci.

[0042] Figure 5D TUNEL staining showed that knockdown of lncATRIG in T3-U87 cells promoted cell apoptosis.

[0043] Figure 5E Statistical graph showing the quantitative analysis of TUNEL-positive cells after 48 h of TMZ treatment.

[0044] Fig. 6A The growth of tumors in the brains of mice transplanted with TMZ-resistant U87 cells infected with lncATRIG inhibitors detected by the small animal in vivo imaging system under the action of the drug.

[0045] Figure 6B Tumor growth curve plotted as grayscale value of tumor luminescence image.

[0046] Figure 6C The body weight change curves of mice in different treatment groups.

[0047] Fig.6D The figure shows the survival curves of mice in different treatment groups. Compared with other groups, the overall survival time of mice after knockdown of lncATRIG and TMZ treatment was prolonged. DETAILED DESCRIPTION

[0048] The embodiments of the present invention will be described in detail and comprehensively below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0049] In recent years, non-coding RNA, especially long non-coding RNA (lncRNA), as an important molecule for regulating gene expression, has gradually been found to be closely related to the occurrence and development of cancer. LncRNA is a class of RNA molecules with a length of more than 200 nucleotides but no protein encoding. It can affect gene expression and cell function through various mechanisms, including interactions with DNA, RNA and proteins. Many studies have shown that lncRNA plays a key role in cancer proliferation, invasion, metastasis and drug resistance. For example, some lncRNAs mediate cancer cells' tolerance to chemotherapeutic drugs by regulating DNA repair pathways, apoptosis signals or tumor stem cell characteristics. Given the important role of lncRNA in drug resistance, intervention methods targeting lncRNA have gradually become a research hotspot for overcoming cancer drug resistance.

[0050] Through high-throughput transcriptome sequencing analysis, researchers have discovered lncRNAs associated with drug resistance in a variety of cancers in recent years, but the non-coding RNAs that play a key role in glioma chemoradiotherapy resistance remain to be explored.

[0051] Temozolomide (TMZ) is the most commonly used chemotherapy drug in the current standard treatment of glioblastoma, usually used in combination with radiotherapy. However, TMZ resistance is common in clinical practice and is an important cause of treatment failure and tumor recurrence. However, the mechanism of TMZ resistance is complex, and the main research involves enhancement of DNA repair mechanisms, gene mutations related to DNA repair, changes in the tumor microenvironment, and other aspects. The regulatory mechanism involving non-coding RNA is also unclear.

[0052] U87MG cell line is the most commonly used human glioblastoma cell line and is widely used in glioma research, including drug screening, gene function analysis and molecular mechanism exploration. Based on this, the present invention identified a new long non-coding RNA in temozolomide-resistant glioma cell line for the first time, named lncATRIG (Long Noncoding RNAs Associated with TMZ-Resistance in Glioma). By constructing an in vivo mouse resistance screening model, the lncRNA was isolated from TMZ-resistant glioma cells. The lncATRIG was abnormally highly expressed in TMZ-resistant cells, suggesting that it may play an important role in the resistance mechanism of glioma.

[0053] Further experiments showed that specific knockdown of lncATRIG expression can significantly inhibit the proliferation of drug-resistant glioma cells and enhance their sensitivity to TMZ. In addition, the inhibition of lncATRIG can also promote the DNA damage response of cells under the action of temozolomide and accelerate the apoptosis of tumor cells. Therefore, lncATRIG is not only an important regulatory factor of glioma resistance, but also a potential therapeutic target. Inhibitors targeting lncATRIG are expected to become a new therapy for the treatment of TMZ-resistant gliomas, bringing new treatment hope to patients with recurrent gliomas.

[0054] The present invention mainly uses commercial U87MG cell lines and other human commercial glioma cell lines. The experimental methods used in the present invention are mostly conventional laboratory molecular biology methods, and the construction of animal tumor models is also a mature technology used in laboratories.

[0055] Material:

[0056] 1. Human glioma cell lines U87MG (U87 for short, Catalog No.: HTB-14) and LN229 (Catalog No.: CRL-2611) were purchased from the American Type Culture Collection (ATCC). Human glioma cell line U251 (Catalog No.: 08061901) was purchased from Sigma-Aldrich.

[0057] 2. High-glucose DMEM medium (Cat. No.: 11966025), Opti-MEM medium for transfection (Cat. No.: 31985070), penicillin-streptomycin (10,000U / ml, Cat. No.: 15140122), 0.25% trypsin (Cat. No.: 25200072) and Lipofectamine TM 2000 transfection reagent (Cat. No.: 11668027) was purchased from Invitrogen (Shanghai) Trading Co., Ltd.

[0058] 3. Test kit:

[0059] RACE amplification kit SMART 5'RACE&3'RACE (Cat. No. 634858) was purchased from Bio-Tech (Beijing) Co., Ltd. NorthernMax TM The kit (Cat. No.: AM1940) was purchased from Invitrogen (Shanghai) Trading Co., Ltd. The TUNEL apoptosis detection kit (Cat. No.: C1098) was purchased from Shanghai Biotech Co., Ltd. The CCK8 detection kit (Cat. No.: 11203ES08) was purchased from Yisheng Biotech (Shanghai) Co., Ltd.

[0060] 4. Chemical reagents

[0061] Temozolomide (Cat. No.: 85622-93-1), hygromycin B (Cat. No.: 31282-04-9) and crystal violet (Cat. No.: C3886) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0062] 5. Packaging vector plasmids pMD.2G (#12259) and psPAX2 (#12260) were purchased from Addgene, Inc. TRIZOL REAGENT (Cat. No. 15596026) was purchased from Invitrogen (Shanghai) Trading Co., Ltd.

[0063] 6. VivoGlo substrate for in vivo imaging detection TM Luciferin (Cat. No.: P1041) was purchased from Promega (Beijing) Biotechnology Co., Ltd.

[0064] 7. Experimental mice: Balb / c nude mice, male, aged 4-6 weeks, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. In a sterile animal room, at a constant temperature of 25-27°C and a constant humidity of 45-50%, placed in a laminar flow cleanroom, 3-5 nude mice were raised in each cage.

[0065] The animal experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the Institute of Biophysics, Chinese Academy of Sciences and were approved by the Animal Ethics Committee and Use Committee.

[0066] instrument:

[0067] 1. Surgical instruments were purchased from Shanghai Mingyuan Industrial Co., Ltd. Suture needles and threads were purchased from Shanghai Pudong Jinhuan Medical Supplies Co., Ltd.

[0068] 2. The small animal in vivo imaging system (IVIS Spectrum) was purchased from PerkinElmer, USA.

[0069] 3. The mouse lightweight brain stereotaxic apparatus (Cat. No. 68805) was purchased from Shenzhen Ruiwode Life Science Co., Ltd.

[0070] Example 1: Preparation of T3-U87 cells

[0071] 1) Mice were selected from male BALB / c nude mice aged 4 to 6 weeks and were raised under standard pathogen-free conditions, with a 12-h light-dark cycle, a temperature of 20-22°C, and a humidity range of 40-50%. All animal experiments were performed under sodium pentobarbital anesthesia, and all measures were based on minimizing animal suffering and were approved by the Animal Experimental Ethics Committee (IACUC) of the Institute of Biophysics, Chinese Academy of Sciences.

[0072] 2) Construction of luciferase expression vector and U87 cells expressing luciferase

[0073] Using the luciferase reporter vector pGL3.0-basic vector (product number: 3666650) purchased from Promega as a template, the luciferase fragment was cloned into the lentivirus (pLenti) system (Addgene, #658-5) by PCR amplification to construct the lentivirus expression luciferase plasmid plenti-CMV-luciferas. The constructed lentivirus expression plasmid pLenti-CMV-luciferase and auxiliary packaging vector plasmid pMD.2G, psPAX2 were extracted with high purity and endotoxin-free plasmid mini-preparation kit (product number: DP118, Tiangen Biochemical Technology (Beijing) Co., Ltd.) and lipofectamine TM 293T cells were transfected with 2000 transfection reagent and lentivirus expressing pLenti-CMV-luciferase was packaged.

[0074] U87 cells were collected at 5×10 5 The cells were seeded at a density of 10 cells / well in a six-well plate and incubated at 37°C with 5% CO 2 Cultured in an incubator. After 18 hours, an appropriate amount of virus was added according to the measured virus titer, and replaced with serum-containing DMEM cell culture medium after 24 hours. After 2 to 3 days of viral infection, hygromycin B was added at a final concentration of 50 μg / ml for resistance screening for 3 days. After testing, a human glioma cell line U87-luc that stably expressed luciferase was obtained. This cell line can be amplified as a P0 generation cell for the next animal experiment.

[0075] 3) Orthotopic brain transplantation of tumor cells:

[0076] After the mouse was anesthetized with isoflurane inhalation anesthesia machine, the animal was fixed to the brain positioning device to keep the head immobile. The skin outside the skull was cut longitudinally with a scalpel to find the bregma point and the needle was inserted at the right anterior temporal side. The needle of a 5 ml syringe was used to rotate and pierce the hole to destroy the skull layer. 5×10 5 U87-luc cells (P0) were resuspended in 10 μl saline, and cells were aspirated with a 10 μl microsyringe. The injection needle was inserted about 2 mm from the right anterior temporal side of the nude mouse. The injection needle was fixed and cells were injected at a speed of 1 μl per minute. After the injection was completed, the needle was slowly withdrawn and the skin was sutured. After the animal woke up, it was sent back to the animal room.

[0077] On day 7 after orthotopic cell transplantation, in vivo imaging of mouse brain xenograft tumors was performed using IVIS Spectrum (PerkinElmer, USA) to confirm successful tumor implantation.

[0078] 4) Seven days after successful orthotopic transplantation of the tumor in the mouse brain, oral DMSO or temozolomide (TMZ) treatment was started at 50 mg / kg / day, with DMSO as the negative control. The treatment regimen was 5 days of treatment per week, 2 days of drug withdrawal, and continued for 3 to 4 weeks. Six mice were taken from each treatment group, and the treatment was repeated three times in total. The treatment cycle of drug administration-drug withdrawal cycle was used to simulate the treatment cycle of tumor chemotherapy drugs, making the cell resistance process closer to the actual clinical situation.

[0079] 5) Separation of tumors and tumor cells: After completing a treatment cycle, the mice were killed and the intact xenografted gliomas were peeled off for cell extraction. First, the tumor was mechanically separated and cut into 1 cubic millimeter tissue fragments, then washed with cold PBS containing penicillin (500U / ml), digested with 0.05% trypsin and DNase (1mg / ml) at 37°C for 15 minutes, and intermittently shaken to obtain a cell suspension. The cells were further filtered through a 100μm filter membrane (Life Science, 352360) and centrifuged at 300g per minute for 5 minutes at 4°C. The resulting cell pellet was resuspended and inoculated into a 6-well plate, completing the first round of in vivo screening under TMZ drug pressure, and the first generation of drug-resistant tumor cells obtained was named T1-U87.

[0080] 6) The T1-U87 obtained above was transplanted back into the brain of another batch of mice according to the above steps 1)-3), and the mice were treated with TMZ or DMSO at the same dose and treatment cycle and then killed; the tumor and tumor cells were separated to obtain the second-generation drug-resistant tumor cells, named T2-U87.

[0081] 7) The T2-U87 obtained above was transplanted back into the brain of another batch of mice according to the above steps 1)-3), and the mice were treated with TMZ or DMSO at the same dose and treatment cycle and then killed; the tumor and tumor cells were separated to obtain the third-generation drug-resistant tumor cells, named T3-U87.

[0082] T3-U87 were cultured in DMEM medium containing 10% FBS, 1% penicillin-streptomycin and 10 μM TMZ for subsequent experiments.

[0083] Example 2: Basic feature analysis of lncATRIG.

[0084] 1. RACE (rapid cDNA end amplification) identification of the full-length sequence of lncATRIG

[0085] As a new long non-coding RNA, in order to obtain the full-length sequence of lncRNA, the RNA sequence of lncATRIG and the accurate location coordinates on the chromosome were first identified by RACE experiment. The experiment mainly used cDNA amplificationkits for 5'and 3'RACE kit (Cat. No.: 634858). The specific steps were carried out according to the kit instructions, which are briefly described as follows:

[0086] (1) Extraction of RNA

[0087] First, total RNA was extracted from U87 cells using the conventional TRIZOL method according to the instruction manual. During extraction, it was necessary to ensure that the RNA was of high quality and free of degradation in order to obtain accurate results.

[0088] (2) Reverse transcription to generate cDNA

[0089] 3'RACE: Reverse transcription of mRNA with a poly(A) tail is performed using a specific primer containing poly(T) to generate cDNA with a known 3' sequence. Reverse transcriptase reverse transcribes mRNA into single-stranded cDNA.

[0090] 5'RACE: Use a gene-specific primer (GSP) for reverse transcription to synthesize cDNA from the 3' end of mRNA. Then, a poly(C) or other specific sequence is added to the 5' end of the cDNA through tailing reaction to facilitate subsequent amplification.

[0091] (3) PCR amplification

[0092] 3'RACE: Using the known 3' terminal sequence (poly(A) tail) and specific primers of the target gene, a specific cDNA sequence is amplified by PCR to obtain the 3' end amplification product.

[0093] 5'RACE: Use universal primers and GSP to perform PCR amplification on the 5' cDNA with the tail added to obtain the amplified product at the 5' end.

[0094] (4) Second round of PCR

[0095] In order to improve the specificity of amplification, a second round of PCR is often required. This round of PCR uses nested primers to further amplify the target fragment and reduce the generation of non-specific products.

[0096] (5) Detection and sequencing

[0097] The amplified product is tested by gel electrophoresis to confirm whether the size of the amplified fragment is in line with the expected size. The amplified product can be purified and sequenced to determine the complete 5' and 3' end sequences.

[0098] Example 2: The basic characteristics of the long non-coding RNA lncATRIG were obtained and assembled using bioinformatics methods through RNA sequencing (RNA-seq) and transcriptomics analysis. The full-length cDNA sequence of lncATRIG, 3837 nt, was obtained through RACE experiments and Sanger sequencing. The results are as follows Figure 1A to Figure 1B Among them, the cDNA sequence of LncATRIG is shown in Seq ID No.1 (3837nt):

[0099]

[0100] like Figure 1A The agarose gel electrophoresis of the PCR products of the 5'RACE (left) and 3'RACE (right) experiments of lncATRIG is shown. The transcription start and end sites of lncATRIG were amplified and identified by the 5'RACE and 3'RACE methods, showing the localization coordinates and sequence of lncATRIG, confirming the existence of its full-length transcript. Analysis of the RACE experiment and the previous experimental RNA-seq combined with histone ChIP-seq data determined that lncATRIG is located on human chromosome 6, spanning the 6p22.3 to 6q27 region, with a total length of 3,837 bp, as shown in Figure 1B The results of transcriptome sequencing and histone antibody chromatin immunoprecipitation DNA sequencing data analysis are shown, showing the visualization of the gene region of lncATRIG on chromosome VI. Figure 1B It can be seen that the expression peak of lncATRIG gene analyzed by RNA-seq shows that the expression of lncATRIG is significantly increased in temozolomide-resistant brain glioma cells T3-U87. It was used to verify the transcriptional activity of lncATRIG and detect key histone modification markers. Figure 1B It can be seen that the lncATRIG gene region has a high peak overlap accompanied by acetylation of K27 on histone H3 (H3K27ac) and trimethylation of K4 on histone H3 (H3K4me3) modifications (H3K27ac and H3K4me3 are usually associated with enhancers and promoters of active genes), which indicates that the region has an active transcriptional state, and also defines the position of the enhancer and promoter regions of the lncATRIG gene in these two overlapping modification peaks.

[0101] 2. Northern blot verification of lncATRIG expression level and transcript size

[0102] Northern blot is a classic RNA detection and quantification technique that detects the presence and expression level of specific RNA through RNA electrophoresis, membrane transfer, and specific probe hybridization. The main steps are briefly described as follows:

[0103] The Northern Blot experiment in this example uses NorthernMax TMThe kit (Thermo Fisher Scientific, AM1940) was used according to the instructions of the kit. First, the specific DNA probe was transcribed in vitro using the RiboMAX large-scale RNA in vitro transcription kit (Promega, P1300) to generate a biotin-labeled antisense probe. After RNA from U87 or T3-U87 cells was separated and electrophoresed on a denaturing PAGE gel, the RNA was transferred to a Brightstar-Plus nylon membrane using the siphon method. The membrane was gently rinsed with water, and the nylon membrane was ultraviolet-crosslinked using the HL-2000HybriLinker (UVP). The hybridization and washing steps were performed at 42°C. The hybridization probe PCR primer sequences are shown in Table 1:

[0104] Table 1 Hybridization probe PCR primer sequences

[0105]

[0106] After hybridization, the membrane was incubated at room temperature with SuperBlock containing RNase inhibitors TM Blocking buffer (ThermoScientific, EO0384) for 1 hour, and then use 800CW Streptavidin (Li- ) secondary antibody detection reagent (VWR, 102673-342) for 1 hour at room temperature. Finally, the images were taken using Typhoon Biomolecular Imager.

[0107] Northern blot results are as follows Figure 1C As shown, Figure 1C The expression abundance of the full-length transcript of lncATRIG RNA in U87 and T3-U87 cells. Figure 1C The left figure shows the results of Northern blot transfer and slide development. It can be seen from the figure that the bands of lncATRIG RNA transcripts can be detected in both U87 and drug-resistant T3-U87 cells, and the expression level of lncATRIG in drug-resistant U87 cells is significantly higher than that in primary U87 cells according to the grayscale analysis of the bands. Signal bands corresponding to 28S and 18S rRNA are shown. Figure 1C The right side is the formaldehyde denaturing agarose gel electrophoresis of RNA in the Northern blot experiment, which shows the positions of the bands corresponding to 28S and 18SrRNA after the total RNA of U87 nuclear drug-resistant cells T3-U87 cells was run on the gel. In RNA electrophoresis, the size of the band can be roughly determined based on the size of the ribosomal RNA. The size of 28S RNA is generally 5kb, and the size of 18S RNA is generally 2kb. Figure 1CThe gel electrophoresis image on the right shows that the RNA band size of lncATRIG is between 28s and 18s, proving that the size of lncATRIG RNA is approximately below 4kb, which is basically consistent with the previous RACE experimental results (3.8kb).

[0108] 3. Single-molecule fluorescence in situ hybridization (smFISH) assay to detect lncATRIG cellular localization

[0109] FISH is a technology that can detect the location of RNA in situ in cells or tissues. By hybridizing fluorescently labeled probes with target RNA, the distribution of RNA in cells or tissues can be visually observed. The main steps include: fixing the tissue or cells, then hybridizing the fluorescently labeled lncATRIG probe or NEAT1 probe with the target RNA, and after washing to remove non-specific binding, using a fluorescence microscope or confocal microscope for observation and imaging. It is a common and mature positioning technology. The FISH probes for lncATRIG and NEAT1 were synthesized by Beijing Deao Biotechnology Co., Ltd.

[0110] The localization of lncATRIG in cells was detected by smFISH. Figure 1D As shown, Figure 1D This is the localization image of lncATRIG RNA in human glioma cell lines analyzed by single-molecule fluorescence in situ hybridization. Figure 1D It can be seen that lncATRIG is co-localized with NEAT1 (a known nuclear non-coding RNA) in the cell nucleus, further illustrating that lncATRIG is mainly located in the cell nucleus.

[0111] from Figures 1A to 1D It can be seen that the basic characteristics of lncATRIG were fully elucidated through chromosome localization, histone modification, RACE amplification and cell localization experiments, laying the foundation for subsequent studies on the function of lncATRIG in temozolomide resistance.

[0112] Example 3: Expression of lncATRIG in normal tissues, glioma tissues and GBM cell lines

[0113] 1. Digital PCR (Digital PCR) detects lncRNA copy number.

[0114] 1. Sample Preparation

[0115] RNA extraction: Total cellular RNA was extracted from tumor and glioma cell samples such as U87, HeLa, 293T, LN229, and U251 using the TRIZOL method. Genomic DNA was removed using DNase and then stored at -80°C for later use.

[0116] Reverse transcription: The extracted RNA was reverse transcribed into cDNA using MMLV reverse transcriptase.

[0117] 2.dPCR Reaction Preparation

[0118] Primer design: The specific primer sequences designed for the target lncRNA are:

[0119] Upstream primer lncATRIG qPCR-F (Seq ID No. 12): 5′-TGGTTGCAAGCTTAGGGGAG-3′;

[0120] Downstream primer lncATRIG qPCR-R (Seq ID No. 13): 5′-GCTTGTTAACGCCACCTTGG-3′;

[0121] The internal reference gene ACTIN was selected as the control group, and the primer sequences were:

[0122] ACTIN-qF (Seq ID No. 14): 5'-CACCATTGGCAATGAGCGGTTC-3';

[0123] ACTIN-qR (Seq ID No. 15): 5'-AGGTCTTTGCGGATGTCCACGT-3'.

[0124] 3. Digital segmentation

[0125] The dPCR instrument uses Bio-Rad's digital PCR instrument to divide the PCR reaction mixture into thousands or even millions of tiny reaction units (such as droplets or nanopores), each of which may contain 0 or 1 target molecule. PCR amplification is performed using SYBR Green dye-labeled PCR enzyme.

[0126] 4. Results Analysis

[0127] By analyzing the fluorescence signal of each unit, dPCR can directly count the number of positive units containing amplified products and the number of negative units that have not been amplified. Calculate the copy number: The absolute copy number of lncRNA in the initial sample is calculated by the ratio of positive units to total units and the Poisson distribution.

[0128] 5. Data Analysis

[0129] The obtained data were compared with the appropriate standard group, the absolute copy number of lncATRIG in the samples was calculated, and statistical analysis was performed. The results are shown in Figure 2A shown.

[0130] from Figure 2AIt can be seen that there are approximately 1000 copies of lncATRIG in T3-U87 cells, while there are only 200 copies in U87 cells, indicating that the RNA copy number of lncATRIG is increased in drug-resistant glioma cell lines, and the expression level is higher than that in sensitive glioma cell lines (LN229, U251) and other tumor cell lines (control group: HeLa, 293T).

[0131] 2. Detection of lncATRIG expression in normal tissues and glioma tissues by quantitative PCR

[0132] 1. Glioma tissue:

[0133] The present invention uses 15 cases of low-grade glioma (LGG) and glioblastoma (GBM), all of which are derived from surgical resection tissues of patients with confirmed glioma in Beijing Tiantan Hospital. Before obtaining the tissues, informed consent was obtained from the patients, and the research use content was informed, and the tissues were approved by the Ethics Committee of Tiantan Hospital.

[0134] 2. Extraction of total RNA from tissues: Trizol reagent was used to extract tumor tissues from 15 LGG and GBM patients, and 2 μg was taken for reverse transcription into cDNA for fluorescence quantitative PCR (RT-qPCR) analysis.

[0135] 3. RT-qPCR: performed using SYBR Green Realtime PCR Master Mix (Yeasen, 10137ES).

[0136] The detection primers used were lncATRIG upstream primer lncATRIG qPCR-F (Seq ID No. 12): 5'-TGGTTGCAAGCTTAGGGGAG-3' and downstream primer lncATRIG qPCR-R (Seq ID No. 13): 5'-GCTTGTTAACGCCACCTTGG-3'. The internal reference gene ACTIN was used as a negative control. The ACTIN upstream primer sequence (and downstream primer sequence were the same as those used in digital PCR. According to the experimental results, qPCR was used for relative quantitative analysis. After three repeated experiments, the relative expression level statistical graph was obtained, as shown in Figure 2. Figure 2B As shown. At the same time, the expression level of CCAT1 (a known long non-coding RNA, associated with colorectal cancer, with high expression abundance in a variety of tumors) in these glioma tissues was detected as a control. The upstream primer of CCAT1 (Seq ID No.16): 5'-GCCGTGTTAAGCATTGCGAA-3'; the downstream primer sequence is (SeqID No.17): 5'-AGAGTAGTGCCTGGCCTAGA-3'. The results are shown in Figure 2C shown.

[0137] from Figure 2B and Figure 2C It can be seen that lncATRIG is more highly expressed in GBM patient samples compared to low-grade glioma (LGG) tissues. In contrast, the highly abundant and oncogenic lncRNA, CCAT1, showed almost no difference. These results suggest that lncATRIG is associated with TMZ resistance and is upregulated in GBM (stage IV resistant recurrent glioma) patient samples.

[0138] 3. Detection of expression and cellular localization in glioma and normal tissues by fluorescence in situ hybridization

[0139] Glioma tissue microarray (Cat. No.: N095Ct01) was purchased from Zhongke Guanghua Xi'an Intelligent Biotechnology Co., Ltd. The microarray contains 85 glioblastomas and 10 normal brain tissues. RNA FISH experiments on glioma tissue microarrays were performed as follows:

[0140] 1. Sample preparation: Dewax the tissue chip (if it is paraffin-embedded tissue), then dehydrate it with gradient alcohol, and finally balance it with PBS.

[0141] 2. Antigen retrieval: Antigen retrieval of tissues by heating or enzyme treatment to improve probe entry and binding efficiency.

[0142] 3. Pretreatment: Use protease to treat the tissue to increase the permeability of the tissue and facilitate the probe to enter the cell.

[0143] 4. Probe hybridization: Add fluorescently labeled specific lncATRIG and NEAT1 probes (the same as the FISH probes used in Example 1) to the sample and perform hybridization overnight at 37°C.

[0144] 5. Washing: Wash several times with hybridization buffer to remove unbound probe.

[0145] 6. Staining: Stain the cell nuclei with nuclear dyes such as DAPI for 15 minutes.

[0146] 7. Sealing: Sealing the slides with anti-fluorescence quenching mounting medium (Vector, H1000).

[0147] 8. Microscope observation: Use Zeiss LSM700 laser confocal fluorescence microscope to observe and take pictures. The results are as follows Figure 2D shown.

[0148] from Figure 2DIt can be seen that compared with the low positive signal in normal brain tissue samples, 90% of GBM tissues were positive for lncATRIG expression and co-localized with NEAT1, and lncATRIG was mainly localized in the cell nucleus. Figure 2E Shown are the statistical results of tissue chip positive cells in normal brain tissue and GBM tissue, with significant differences.

[0149] According to the above results, lncATRIG was verified by the above RACE, NB and FISH experiments and was confirmed to be a new long non-coding RNA gene that had not been discovered before and existed on chromosome 6. lncATRIG was highly expressed in 90% of GBM patient tumor tissues, and the expression was co-localized with NEAT1 (a long non-coding RNA that has been reported to be associated with tumor resistance and highly expressed), indicating that lncATRIG has similar functions to NEAT1 and is also involved in the biological occurrence and drug resistance process of GBM.

[0150] Example 4: The promoting effect of lncATRIG overexpression on temozolomide resistance

[0151] In this example, the method of overexpressing lncATRIG was used to verify its effect in glioma cells, especially its effect on temozolomide (TMZ) resistance, and the cell proliferation and invasion abilities were evaluated by colony formation and invasion experiments.

[0152] Experimental methods:

[0153] 1. Overexpression of lncATRIG in glioma cells

[0154] 1. Cell culture: Three human glioma cell lines, U87, LN229 and U251, were cultured in DMEM medium containing 10% serum.

[0155] 2. Plasmid Construction

[0156] To overexpress lncATRIG, the full-length exon sequence (3,837 nt) was PCR amplified from cDNA of T3-U87 cells and subsequently cloned into pLenti-CMV-GFP-Hygro plasmid (Addgene, #656-4). At the same time, a plasmid containing λ DNA of equal length was also constructed as a negative control.

[0157] PCR primer sequences used:

[0158] ATRIG-full-length PCR upstream primer (Seq ID No.18):

[0159] 5'-CCTCTTCCCCTCCCTTGTC-3'

[0160] ATRIG-full-length PCR downstream primer (Seq ID No.19):

[0161] 5'-TGTAGTCTAATTTATTGTAGCATTTATTCCTG-3'

[0162] λDNA-3800 PCR upstream primer (Seq ID No. 20): 5'-GTTCCTGGGTGACAAGCGTA-3'

[0163] λDNA-3800 PCR downstream primer (Seq ID No. 21): 5′-AAGATGGCTGTAGGGGTTGC-3′.

[0164] 3. Cell Transfection

[0165] Refer to the instructions of Lipo2000 transfection reagent, as briefly described below:

[0166] (1) One day before transfection, U87 cells, LN229 cells or U251 cells were cultured at 3×10 5 / well were seeded into 6-well plates to ensure that the cells were in the logarithmic growth phase on the day of transfection.

[0167] (2) In a sterile tube, mix the required amount of plasmid DNA with serum-free medium (Opti-MEM), 2 μg of plasmid overexpressing ATRIG or λ DNA per well. Add Lipo2000 transfection reagent (at a 1:2 ratio of DNA to serum-free medium), gently invert to mix, and let stand for 5-15 minutes to form a complex.

[0168] (3) Slowly add Lipo2000-DNA complex into the cell culture medium and shake gently to ensure even distribution.

[0169] (4) Place the cells at 37°C and 5% CO 2 After 4-6 hours, the culture medium was replaced with serum-containing medium.

[0170] (5) The status of cells was observed 24 hours after transfection. After 48 hours, the transfection efficiency and gene expression were detected by RT-qPCR method.

[0171] 2. Colony Formation Assay

[0172] (1) Cell inoculation and culture:

[0173] After culturing U87 cells, LN229 cells or U251 cells overexpressing lncATRIG after transfection for 24 hours, the cells were counted and seeded into 6-well plates at 5,000 cells per well to ensure uniform cell seeding.

[0174] (2) Cell treatment and colony formation

[0175] After the cells adhered, 10 μM TMZ or DMSO was added for treatment. After 48 hours, the culture medium was replaced with fresh drug-free culture medium. The cells were cultured for another 10 days. Cell proliferation was observed and colonies visible to the naked eye were formed (one colony usually consisted of more than 50 cells).

[0176] (3) Fixed cells

[0177] Prepare methanol or ice-cold methanol:ethanol (1:1) solution as fixative. Pour off the culture medium, gently wash the cells twice with PBS, and then add the fixative. Fix the cells for 15 minutes at room temperature, aspirate the fixative, and keep the culture plate dry at room temperature.

[0178] (4) Crystal violet staining

[0179] Prepare 0.5% crystal violet staining solution: 0.5g crystal violet is dissolved in 100ml 20% methanol aqueous solution. Add 1ml crystal violet staining solution to the fixed cells and stain for 15 minutes at room temperature. Pour out the staining solution and rinse the culture plate with tap water until the background becomes clear and transparent with no excess dye residue.

[0180] (5) Drying and observation and photography

[0181] The stained culture plate was dried at room temperature, observed under a microscope or with the naked eye, and photographed, and the number of cell colonies formed in each well or dish was counted.

[0182] (6) Data analysis

[0183] Count the number of colonies in each well or culture dish. Calculate the colony formation rate according to the colony formation rate calculation formula: colony formation rate (%) = number of colonies in the experimental group / number of cells inoculated × 100. Compare the colony formation rates of the treatment group and the control group to evaluate the effect of TMZ on cell proliferation ability.

[0184] The results are as follows FIG. 3A to FIG. 3F As shown, λ-DNA and lncATRIG plasmids of the same length (3.8 kb) were overexpressed into U87, LN229 and U251 cells, respectively. The two groups of cells were treated with dimethyl sulfoxide (DMSO, solvent control) and temozolomide (TMZ, chemotherapeutic drug), respectively. In the three human glioma cell lines U87, LN229 and U251, compared with cells overexpressing λ-DNA of the same length (3.8 kb) (control cells, Ctrl), lncATRIG overexpression (ATRIG OE) significantly increased the cell resistance to TMZ. FIG. 3A to FIG. 3FThe results of colony formation experiments showed that after TMZ treatment, the cell line overexpressing lncATRIG retained more cell viability and formed more colonies compared with the control group (Ctrl:λDNA group).

[0185] 3. Transwell assay

[0186] 1. Cell culture:

[0187] U87 cells, LN229 cells, and U251 cells overexpressing lncATRIG after transfection were seeded in culture dishes and cultured to the logarithmic growth phase.

[0188] 2. Transwell chamber preparation: Use a chamber with a pore size of 8 μm and add 500 μl of cell culture medium containing 10% serum to the lower chamber.

[0189] 3. Cell seeding:

[0190] Count the cells and add the cell suspension to the upper chamber, usually 1×10 5 Prepare cell suspension using serum-free medium.

[0191] 4. Place the Transwell chamber in an incubator, usually at 37°C and 5% CO 2 Incubate for 36 hours.

[0192] 5. Fixation and staining:

[0193] After treatment, remove the Transwell chamber, wash the upper and lower chambers with PBS, fix the cells with 4% paraformaldehyde for 10 minutes, and stain with 0.5% crystal violet stain for 15 minutes at room temperature.

[0194] 6. Cleaning and observation analysis:

[0195] Wash with PBS to remove unbound dye. Observe the cells under a microscope and count the number of migrated and invaded cells. Calculate the number of migrated or invaded cells based on the counting results and perform statistical analysis. The results are shown in Figure 3G to Figure 3L shown.

[0196] from Figure 3G to Figure 3L It can be seen that in the cell invasion experiment, lncATRIG overexpression also enhanced the invasion ability of U87 cells, LN229 cells, and U251 cells, especially under TMZ treatment, the invasion index was significantly increased.

[0197] Example 5: Effects of lncATRIG inhibitors on proliferation and invasion of TMZ-resistant U87 cells

[0198] This example mainly evaluates the effect of lncATRIG inhibitor on the proliferation and invasion ability of TMZ-resistant U87 cells, and explores the role of lncATRIG in the drug resistance of glioma cells.

[0199] First, an inhibitor targeting the degradation of ATRIG, namely a locked nucleic acid modified ASO sequence, was designed based on the RNA sequence of the lncATRIG gene. Locked nucleic acid (LNA) is a modified sequence in which the 2' and 4' carbons on part or all of the ribose in LNA are linked together, and is generally found in A-DNA or RNA. This type of nucleic acid can increase the melting temperature (Tm value) of primers or probes and enhance the stability of these experimental substances. The present invention designed three antisense oligonucleotides Seq ID No.2 to Seq ID No.4 targeting the full length of lncATRIG based on the RNA structure of lncATRIG, each with a length of 16nt, and locked nucleic acid and methoxy modification on each nucleotide. All sequences were commissioned to QIAGEN (China) for synthesis.

[0200] The inhibitor sequences targeting lncATRIG are shown in Table 2:

[0201] Table 2: Inhibitor sequences targeting lncATRIG

[0202] name Serial number sequence LNA-1 Seq ID No.2 5'-GAGAGTCCATTAGAGG-3' LNA-2 Seq ID No.3 5'-TAGGTTGCTAGGGAGT-3' LNA-3 Seq ID No.4 5'-TCGAAGTTAATTGAAT-3'

[0203] The TMZ-resistant U87 cell line was cultured to the logarithmic growth phase, and the specific lncATRIG inhibitor was transfected with Lipo. The three LNAs were mixed and 100 pmol was added to 5×10 5 In T3-U87 cells, the expression level of lncATRIG was detected by RT-qPCR 48 h after transfection to determine the knockdown efficiency, e.g. Figure 4A As shown, after transfection with three targeted LNAs, it was called LNA-ATRIG (LNA-NC was the control), and the total RNA of the cells was extracted for RT-qPCR to detect the RNA knockdown efficiency of lncATRIG. The results showed that after LNA-ATRIG transfection, the RNA expression level of lncATRIG in the cell decreased by 85-90%.

[0204] This example then performs a colony formation experiment to evaluate the proliferation ability of the cells. The experimental method is the same as that of Example 3. It mainly includes the following steps: T3-U87 cells after knocking down lncATRIG are cultured in a culture medium containing TMZ, and approximately 1,000 cells are seeded into a 6-well plate per well and cultured for 14 days to form colonies. The culture medium is replaced every few days, and the formation of colonies is observed regularly. After the culture is completed, the cells are fixed with 4% paraformaldehyde, the colonies are stained with crystal violet staining, and the number of colonies is observed and counted under a microscope after washing, as shown in FIG. Figure 4B shown. Figure 4C The results of quantitative analysis of colony numbers are shown. Figure 4B and Figure 4C It can be seen that compared with the control group, the proliferation ability of T3-U87 cells with lncATRIG knockdown was reduced under the action of TMZ, indicating that knocking down lncATRIG has a restoring effect on the drug sensitivity of drug-resistant cells, thereby inhibiting the proliferation of drug-resistant tumor cells.

[0205] In addition, the invasion ability of cells was also evaluated by Transwell assay. The experimental method was the same as in Example 3. T3-U87 cells with lncATRIG knockdown were inoculated into Transwell chambers containing basement membrane coating and cultured for 24 hours. TMZ was added to the lower chamber of Transwell. After treatment, non-invaded cells were removed with a cotton swab and stained with crystal violet, and the number of penetrated cells was counted, as shown in Figure 3. Figure 4D shown. Figure 4E The quantitative analysis results of invasion index are shown. Figure 4D and Figure 4E It can be seen that compared with the control group, the T3-U87 cells with lncATRIG knockdown had reduced cell invasion ability under the action of TMZ, indicating that knocking down lncATRIG has a restoring effect on the drug sensitivity of drug-resistant cells, thereby inhibiting the invasion ability of drug-resistant tumor cells.

[0206] RT-qPCR results showed that the expression level of lncATRIG was significantly reduced, verifying the effectiveness of knockdown. Further analysis of cell proliferation and invasion ability showed that after knocking down lncATRIG, the colony formation ability of T3-U87 cells under TMZ treatment was significantly reduced, and quantitative analysis also confirmed this result, indicating that lncATRIG has a promoting effect on the proliferation ability of glioma cells.

[0207] The results of this example show that lncATRIG plays an important role in the proliferation and invasion of TMZ-resistant U87 cells. Knockdown of lncATRIG significantly inhibited the colony formation and invasion ability of cells, suggesting that it may be used as a potential therapeutic target. By inhibiting the expression of lncATRIG, it may enhance the therapeutic effect of TMZ on glioma and reduce drug resistance. Therefore, intervention measures targeting lncATRIG may help improve the clinical efficacy of TMZ and further explore its potential application in the treatment of glioma.

[0208] Example 6: lncATRIG inhibitors promote TMZ-induced DNA damage and apoptosis and increase TMZ sensitivity

[0209] This example investigated whether lncATRIG inhibitors increase TMZ sensitivity in T3-U87 cells by enhancing TMZ-induced DNA damage and promoting apoptosis.

[0210] The main experimental methods are as follows:

[0211] 1. CCK8 calculation of the IC50 value of cell response to TMZ after knockdown of lncATRIG

[0212] T3-U87 cells were transfected with a specific lncATRIG inhibitor to achieve effective knockdown of lncATRIG. After knockdown, the CCK8 method was used to detect the resistance of cells to TMZ, and the IC50 value was calculated to evaluate the sensitivity of cells to TMZ. Figure 5A The results showed that after knocking down lncATRIG, the drug-resistant cell T3-U87 had a lower half lethal dose of TMZ, and the cell's drug sensitivity to TMZ was significantly improved.

[0213] CCK8 detection of cell viability and IC50 value calculation steps:

[0214] (1) Cell culture:

[0215] The TMZ-resistant cells T3-U87 with lncATRIG knockdown were seeded into 96-well plates at 2000 cells per well and cultured for 24 hours to reach the logarithmic growth phase. U87 cells served as the control group.

[0216] (2) TMZ treatment:

[0217] Prepare a series of drug solutions with different concentrations (usually 600μM, 500μM, 400μM, 300μM, 200μM, 100μM from high to low concentration gradient), and add these different concentrations of TMZ-containing culture medium to different wells of the cell culture plate. Set up 3 replicate wells for each concentration to ensure the reliability of the data. Continue to culture the cells for 48 hours to ensure that the drug can fully exert its effect.

[0218] (3) CCK8 staining:

[0219] CCK8 reagent (Cell Counting Kit-8) was added to each well. Since WST-8 in CCK8 reagent would be reduced to orange-yellow formazan product by dehydrogenase in cells, the optical density (OD) of each well at 450nm wavelength could be measured. The OD value was proportional to the activity of cells.

[0220] (4) Calculate the IC50 values ​​of different cells' responses to TMZ based on the OD values:

[0221] According to the OD values ​​at different drug concentrations, a curve of TMZ concentration and cell viability (ratio of OD value to control group) is drawn. The drug concentration that reduces cell viability by 50%, i.e., IC50 value, is calculated by data fitting (e.g., using nonlinear regression analysis).

[0222] 2. Immunofluorescence analysis of DNA damage marker expression changes

[0223] Immunofluorescence staining was used to analyze the expression of γ-H2AX and RAD51 in TMZ-treated T3-U87 cells after knockdown of lncATRIG. The experimental steps were as follows: cells were fixed for 10 minutes after TMZ treatment. After fixation, cells were rinsed three times with PBS for 5 minutes each, and then the cells were permeabilized on ice for 15 minutes in PBS buffer containing 0.5% Triton X-100. Subsequently, cells were blocked with 1% BSA at room temperature for 15 minutes, and γ-H2AX antibody (Abcam, ab243906) and RAD51 antibody (Abcam, ab133553) were added and incubated in 1% BSA at 4°C overnight. The next day, Alexa Fluor TM The cells were incubated with goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibodies (Invitrogen, A-11012) labeled with 594 and 488 at room temperature for 1 hour in the dark. The cell nuclei were stained with DAPI for 15 minutes. After sealing with antifade mounting medium, the cells were observed with a Zeiss LSM700 laser confocal microscope and immunofluorescence photos were taken.

[0224] 3. TUNEL assay to detect cell apoptosis after knockdown of lncATRIG

[0225] In addition, TUNEL staining was used to evaluate the effect of knocking down lncATRIG on cell apoptosis. In the experiment, T3-U87 cells were stained with TUNEL after 48 hours of TMZ treatment, and the number of TUNEL-positive cells was observed and counted. The quantitative analysis on the right shows the results of TUNEL-positive cells after 48 hours of TMZ treatment.

[0226] The results of CCK8 experiments are as follows Figure 5A As shown in Figure 2, knocking down lncATRIG significantly reduced the resistance of T3-U87 cells to TMZ, and the IC50 value decreased significantly. This indicates that lncATRIG is a key regulatory factor in TMZ resistance, and inhibiting its expression can improve the effect of chemotherapy drugs. Further immunofluorescence experimental results are shown in Figure 2. Figure 5B and Figure 5C As shown in Figure 2, lncATRIG inhibitors promoted DNA damage accumulation by enhancing TMZ-induced γ-H2AX and RAD51 expression. DNA damage is the main mechanism of TMZ-induced cell death. Knockdown of lncATRIG made cells more sensitive to DNA damage, showing its important regulatory role in the DNA repair pathway. At the same time, the TUNEL experiment results were as follows Figure 5D and Figure 5E As shown in Figure 2, knockdown of lncATRIG significantly enhanced the apoptosis rate of cells after TMZ treatment. Apoptosis is a self-destruction mechanism of cells when DNA damage is irreparable. The action of lncATRIG inhibitors exacerbates this process, thereby enhancing the therapeutic effect of TMZ.

[0227] from FIG. 5A to FIG. 5E It can be seen that lncATRIG inhibitors significantly increased the sensitivity of T3-U87 cells to TMZ by enhancing TMZ-induced DNA damage and apoptosis. This indicates that lncATRIG, as an important regulator of TMZ resistance, may become a potential therapeutic target for targeting glioma resistance. In the future, inhibitors targeting lncATRIG may be used to enhance the effect of TMZ chemotherapy, thereby improving the prognosis of glioma patients.

[0228] Example 7: lncATRIG inhibitor promotes the sensitivity of brain transplanted tumors in drug-resistant mice to TMZ

[0229] This example is used to evaluate the growth inhibitory effect of lncATRIG inhibitor on TMZ-resistant U87 cell tumors in a mouse brain transplant model and explore the potential application of lncATRIG in tumor treatment.

[0230] Experimental methods:

[0231] 1. Use BLOCK-iT TMShort hairpin RNAs (shRNAs) were designed by RNAi Designer (https: / / rnaidesigner.thermofisher.com / ). DNA oligonucleotides corresponding to shRNAs and related random sequences were synthesized and cloned individually into pLKO.1-TRC vector (Addgene, #10878).

[0232] 2. Establishment of TMZ-resistant U87 cell line with stable knockdown of lncATRIG

[0233] 3. Establishment of nude mouse brain tumor model.

[0234] 1) Male BALB / c nude mice aged 4 to 6 weeks (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were selected and raised under standard pathogen-free conditions, with a 12-h light-dark cycle, a temperature of 20-22°C, and a humidity range of 40-50%. All animal experiments were performed under sodium pentobarbital anesthesia, and all measures were based on minimizing animal suffering and were approved by the Animal Experiment Ethics Committee (IACUC) of the Institute of Biophysics, Chinese Academy of Sciences.

[0235] 2) Orthotopic transplantation of tumor cells into the brain:

[0236] After nude mice were anesthetized with isoflurane inhalation anesthesia machine, they were fixed to a brain positioner to keep their heads still. The skin outside the skull was cut longitudinally with a scalpel to find the bregma point and the needle was inserted at the right anterior temporal side. The needle of a 5 ml syringe was used to rotate and pierce the skull layer. 5×10 5 U87 cells (P0) stably expressing firefly luciferase were resuspended in 10 μl of saline. The cells were aspirated with a 10 μl microsyringe. The injection needle was inserted about 2 mm from the right anterior temporal side of the nude mouse. The injection needle was fixed and the cells were injected at a speed of 1 μl per minute. After the injection was completed, the needle was slowly withdrawn and the skin was sutured. The animal was sent back to the animal room after it woke up.

[0237] On day 7 after orthotopic cell transplantation, in vivo imaging of mouse brain xenograft tumors was performed using IVIS Spectrum (PerkinElmer, USA) to confirm successful tumor implantation.

[0238] 3) Seven days after successful orthotopic transplantation of the tumor in the mouse brain, oral DMSO (50 mg / kg / day) or TMZ (50 mg / kg / day) treatment was started. The treatment regimen was 5 days per week, followed by 2 days of rest for 3 to 4 weeks. Six mice were selected from each treatment group, and the experiment was repeated three times.

[0239] 4) Bioluminescence imaging was performed on days 7, 14, 21, and 28 of treatment to monitor intracranial tumor growth. At the same time, the weight of mice was recorded every other day, and the data were normalized to the luminescence value detected for each animal at the beginning of treatment.

[0240] During the treatment, the growth of the tumor was monitored using a small animal in vivo imaging system. The imaging results on days 7, 14, 21, and 28 showed that the lncATRIG inhibitor combined with TMZ treatment significantly inhibited the growth of brain tumors compared with the control group. The in vivo imaging data showed that the tumor luminescence signal of mice after lncATRIG knockdown was greatly weakened, indicating that the tumor volume and activity were significantly reduced. Fig. 6A shown. Figure 6B The gray value curve of tumor growth is shown, proving that the tumor growth dynamics are significantly inhibited. In order to evaluate the overall health of mice in different treatment groups, the weight changes of mice were recorded every week during the experiment, such as Figure 6C As shown. Figure 6C It can be seen that there is no significant difference in the weight change of mice in each group, indicating that the lncATRIG inhibitor has no negative impact on the overall health of mice. At the same time, the survival time of mice in different treatment groups was monitored and the survival curve was drawn, such as Fig.6D As shown, the survival rate of mice after lncATRIG knockdown and TMZ treatment was compared with that of other groups. Fig.6D It can be seen that the overall survival time of the mouse group treated with lncATRIG inhibitor and TMZ was significantly prolonged, and the survival rate was higher than that of TMZ alone or the control group.

[0241] from FIG. 6A to FIG. 6D It can be seen that the lncATRIG inhibitor significantly enhanced the inhibitory effect of TMZ on drug-resistant U87 cell brain transplant tumors without affecting the health status of mice. By combining TMZ and lncATRIG inhibitors, tumor growth was effectively inhibited and the survival rate was significantly improved. This illustrates the key role of lncATRIG in glioma resistance. By targeting lncATRIG, it is expected to improve the efficacy of TMZ and provide a new therapeutic strategy for the clinical treatment of drug-resistant gliomas.

[0242] The in vivo experiments in this example confirmed the significant role of lncATRIG inhibitors in enhancing the effect of TMZ. This laid the foundation for subsequent clinical transformation, demonstrated the potential of overcoming drug resistance by regulating non-coding RNA, and is expected to bring longer survival and better prognosis to glioma patients.

[0243] As can be seen from the above embodiments, the present invention obtains the lncRNA molecule lncATRIG, which is closely related to glioma TMZ resistance, through an in vivo mouse resistance screening model and transcriptome sequencing technology, and verifies its high expression in resistant cells and its function of promoting tumor cell proliferation, invasion and resistance through a series of experiments. This provides a new molecular target for the study of drug-resistant gliomas and reveals a new mechanism of glioma resistance. The present invention designs a specific antisense oligonucleotide (ASO) inhibitor for the lncATRIG sequence, which can effectively knock down the expression of lncATRIG, thereby significantly inhibiting the proliferation of glioma cells and enhancing the chemotherapy effect of TMZ, which is particularly suitable for patients with drug-resistant gliomas. This inhibitor provides a precise molecular tool for future personalized treatment plans. The inhibitor of lncATRIG can not only overcome TMZ resistance, but also enhance the sensitivity of tumor cells to TMZ, further accelerate the DNA damage response caused by chemotherapy drugs, and increase the apoptosis rate of tumor cells. This brings new treatment options for patients with recurrent gliomas and fills the gap of the lack of effective sensitizers in the clinic. At the same time, it creates a new path for targeting lncRNA to treat tumors. Its broad application prospects are not limited to gliomas, and can be extended to drug resistance problems in other types of cancer in the future. Through the present invention, the inhibitor of lncATRIG is expected to provide a new molecular targeted therapy for tumor patients, providing a solid scientific basis for overcoming the clinical problem of glioma drug resistance.

[0244] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention and these improvements should also be regarded as the scope of protection of the present invention.

Claims

1. A lncATRIG inhibitor, characterized in that It includes one or more of the sequences LNA-1, LNA-2, and LNA-3 shown below; wherein the sequence of LNA-1 is shown in Seq ID No.2; the sequence of LNA-2 is shown in Seq ID No.3; the sequence of LNA-3 is shown in Seq ID No.4; and the cDNA sequence of lncATRIG is shown in Seq ID No.

1.

2. The lncATRIG inhibitor according to claim 1, characterized in that Locked nucleic acid and methoxy modification were performed on each nucleotide of the LNA-1, the LNA-2 and the LNA-3.

3. Use of the lncATRIG inhibitor as claimed in claim 1 or 2 in the preparation of a medicament for inhibiting the proliferation of brain glioma cells.

4. Use of the lncATRIG inhibitor as claimed in claim 1 or 2 in the preparation of a temozolomide sensitizing drug.

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