An lncatrigen inhibitor and its use in the preparation of a drug for treating glioma

Abstract

The application discloses a lncATRIG inhibitor and application thereof in preparation of a drug for treating glioma. The lncATRIG inhibitor comprises one or more of sequences LNA-1, LNA-2 and LNA-3 shown in the following formula; wherein the sequence of the LNA-1 is shown in Seq ID No. 2; the sequence of the LNA-2 is shown in Seq ID No. 3; the sequence of the LNA-3 is shown in Seq ID No. 4; and the cDNA sequence of the lncATRIG is shown in Seq ID No. 1. The lncATRIG inhibitor provided by the application can inhibit the proliferation of brain glioma cells and promote temozolomide sensitization.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor molecular biology, and relates to a long non-coding inhibitor and application thereof, in particular to a lncATRIG inhibitor and application thereof in preparation of a glioma treatment drug and a chemotherapy drug sensitizer. BACKGROUND

[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. According to histological morphology and molecular characteristics, the World Health Organization (WHO) classifies glioma into types I to IV, among which types I and II are referred to as low-grade glioma (LGG), and types III to IV include glioblastoma (GBM), anaplastic astrocytoma and anaplastic oligodendroglioma. Due to the extremely high drug resistance of glioma, high recurrence rate and serious impact on the quality of life of patients, these factors greatly shorten the survival time of patients, and therefore types III and IV glioma are often the focus of research. The incidence of low-grade glioma is low, accounting for only 15% of the total number of glioma, and the treatment effect is good and the prognosis is better through surgical resection, radiotherapy and chemical drug treatment, and about 47% of patients can survive for more than ten years. In contrast, malignant glioma is one of the brain tumors with extremely high malignancy, although its total incidence is only 5.55 / 10 million, but the survival rate of patients is only one-third. The average survival time of patients without intervention is three months, and the survival time after treatment is 12 to 15 months, and the longest is not more than five years. Malignant tumors have become the main reason for premature death and shortened life expectancy in many countries.

[0003] Current tumor treatment strategies include surgery, chemotherapy, radiotherapy and immunotherapy, but still face 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 for treating GBM. However, long-term use of temozolomide can easily lead to drug resistance, leading patients to face limited treatment options after recurrence, or even no available drugs. The complexity of temozolomide resistance has brought great challenges to the treatment of glioma, and it is urgent to find new molecular targets and treatment strategies.

[0004] Drug resistance is the main factor limiting the efficacy of temozolomide during the treatment. Glioma cells develop drug resistance to temozolomide through various mechanisms, among which the most common one is the enhancement of DNA repair mechanism. 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 antitumor effect of temozolomide. Glioma cells with high expression of MGMT have lower sensitivity to temozolomide, indicating a poor prognosis for patients. In addition, temozolomide resistance also involves the regulation of multiple aspects such as DNA mismatch repair system, apoptosis signaling pathway and tumor stem cell characteristics. The complex mechanism of drug resistance prompts researchers to find new drug resistance related factors in order to develop more effective treatment programs. SUMMARY

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

[0006] Another purpose of the present application is to provide the application of the lncATRIG inhibitor.

[0007] In order to achieve the above purpose, the present application provides a lncATRIG inhibitor, which comprises one or more of the following sequences LNA-1, LNA-2, LNA-3; wherein the sequence of the LNA-1 is shown in Seq ID No. 2; the sequence of the LNA-2 is shown in Seq ID No. 3; the sequence of the LNA-3 is shown in Seq ID No. 4; the cDNA sequence of the lncATRIG is shown in Seq ID No. 1.

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

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

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

[0011] The present application has the following beneficial effects:

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

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

[0014] Figure 1B Visualization of the genomic region of lncATRIG on chromosome 6.

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

[0016] Figure 1D Images of the localization of lncATRIG RNA in human glioma cell lines analyzed by single molecule fluorescent in situ hybridization.

[0017] Figure 2A Copy number analysis of LncATRIG in different tumor cells.

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

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

[0020] Figure 2D Expression of LncATRIG in human glioma tissue chip analyzed by RNA FISH.

[0021] Figure 2E Statistical plot of tissue chip positive cells in normal brain tissue and GBM tissue.

[0022] Figure 3A Comparison photo of overexpression of lncATRIG promoted the colony formation ability of U87 cells in the presence of TMZ.

[0023] Figure 3B Statistical analysis results plot of colony formation ability after overexpression of lncATRIG.

[0024] Figure 3C Comparison photo of overexpression of lncATRIG promoted the colony formation ability of LN229 cells in the presence of TMZ.

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

[0026] ​Figure 3E The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0027] Figure 3F The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ. Figure 3E The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0028] Figure 3G The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0029] Figure 3H The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ. Figure 3G The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0030] Figure 3I The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0031] Figure 3J The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ. Figure 3I The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0032] Figure 3K The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0033] Figure 3L The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ. Figure 3K The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0034] Figure 4A The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0035] Figure 4B The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0036] Figure 4C The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ. Figure 4B The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0037] Figure 4D The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0038] Figure 4E The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ. Figure 4D The comparison photo of the colony formation ability of U251 cells promoted by overexpression of lncATRIG in the presence of TMZ.

[0039] Figure 5AStatistical graph of CCK8 assay to detect the cell resistance to TMZ (IC50) after knocking down lncATRIG in T3-U87 cells.

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

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

[0042] Figure 5D TUNEL staining shows that knocking down lncATRIG in T3-U87 cells promotes apoptosis.

[0043] Figure 5E Statistical graph of quantitative analysis of TUNEL positive cells after 48 hours of TMZ treatment.

[0044] Figure 6A Tumor growth under the action of drugs in mice transplanted with TMZ-resistant U87 cells infected with lncATRIG inhibitors detected by a small animal live imaging system.

[0045] Figure 6B Tumor growth curve plotted from the gray value of the tumor luminescence image.

[0046] Figure 6C Body weight change curve of mice in different treatment groups.

[0047] Figure 6D Survival curve of mice in different treatment groups. Compared with other groups, the overall survival time of mice after knocking down lncATRIG and TMZ treatment is prolonged. DETAILED DESCRIPTION

[0048] The embodiments of the present application will be described in detail below, so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the protection scope of the present application can be more clearly defined.

[0049] In recent years, non-coding RNA, especially long non-coding RNA (lncRNA), has been found to be closely related to the occurrence and development of cancer as an important molecule for regulating gene expression. LncRNA is a kind of RNA molecule with a length of more than 200 nucleotides but not encoding protein, which can affect gene expression and cell function through various mechanisms, including interaction with DNA, RNA and protein. Many studies have shown that lncRNA plays a key role in the proliferation, invasion, metastasis and drug resistance of cancer. For example, certain lncRNAs mediate the resistance of cancer cells to chemotherapeutic drugs by regulating DNA repair pathways, apoptosis signals or tumor stem cell properties. Given the important role of lncRNA in drug resistance, intervention targeting lncRNA has gradually become a research hotspot for overcoming cancer drug resistance.

[0050] Through high-throughput transcriptome sequencing analysis, researchers have found lncRNAs related to drug resistance in various cancers in recent years, but the non-coding RNA that plays a key role in radiotherapy and chemotherapy resistance of glioma still needs to be explored.

[0051] Temozolomide (TMZ) is the most commonly used chemotherapeutic drug in the current standard treatment regimen for glioblastoma, and is usually used in combination with radiotherapy. However, TMZ resistance is prevalent in the clinic and is an important reason for treatment failure and tumor recurrence. However, the mechanism of TMZ resistance is complex, and the main research involves the enhancement of DNA repair mechanism; gene mutations and changes in tumor microenvironment related to DNA repair, and the regulatory mechanism involving non-coding RNA is not clear.

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

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

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

[0055] Materials:

[0056] 1. Human glioma cell lines U87MG (abbreviated as U87, item number: HTB-14) and LN229 (item number: CRL-2611) were purchased from American Type Culture Collection (ATCC). Human glioma cell line U251 (item number: 08061901) was purchased from Sigma-Aldrich Company.

[0057] 2. High-glucose DMEM medium (item number: 11966025), Opti-MEM medium for transfection (item number: 31985070), penicillin-streptomycin (10,000 U / ml, item number: 15140122), 0.25% trypsin (item number: 25200072) and Lipofectamine TM 2000 transfection reagent (item number: 11668027) were purchased from Thermo Fisher Scientific (Shanghai) Trading Co., Ltd.

[0058] 3. Kits:

[0059] RACE amplification kit SMART 5' RACE & 3' RACE (item number: 634858) was purchased from Bioduro Biotech (Beijing) Co., Ltd. NorthernMax TM Kit (item number: AM1940) was purchased from Thermo Fisher Scientific (Shanghai) Trading Co., Ltd. TUNEL cell apoptosis detection kit (item number: C1098) was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. CCK8 detection kit (item number: 11203ES08) was purchased from Yixing Biotechnology 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 vectors plasmid pMD.2G (#12259), psPAX2 (#12260) were purchased from Addgene, USA. TRIZOL REAGENT (Cat. No. 15596026) was purchased from Invitrogen (Shanghai) Trading Co., Ltd.

[0063] 6. VivoGlo Luciferin (Cat. No. P1041) was purchased from Promega (Beijing) Biotech Co., Ltd. TM Luciferin (Cat. No. P1041) was purchased from Promega (Beijing) Biotech Co., Ltd.

[0064] 7. Experimental mice: Balb / c nude mice, male, 4-6 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. In a sterile level animal room, the temperature was kept at 25-27℃, and the humidity was kept at 45-50%. Three to five nude mice were raised in each cage.

[0065] The operation of animal experiments strictly followed the guidelines for the management and use of experimental animals of the Institute of Biophysics, Chinese Academy of Sciences, and was approved by the Animal Ethics Committee and the Use Committee.

[0066] Instruments:

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

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

[0069] 3. Mouse light portable brain stereotaxic apparatus (Cat. No. 68805) was purchased from Shenzhen Ruivode Life Science and Technology Co., Ltd.

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

[0071] 1) Mice were selected as 4-6 week old male BALB / c nude mice raised under standard pathogen-free conditions, with a 12 hour light-dark cycle, maintaining the temperature at 20-22℃, and the humidity range of 40-50%. All animal experiments were performed under pentobarbital sodium anesthesia, and all measures were based on the principle of minimizing animal suffering, and were approved by the Institute of Biophysics, Chinese Academy of Sciences Animal Experiment Ethics Committee (IACUC).

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

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

[0074] U87 cells were seeded in a six-well plate at a density of 5x10 5 cells / well, and cultured in a 37°C, 5% CO2 incubator. After 18 hours, the appropriate amount of virus was added according to the determined virus titer, and after 24 hours, the medium was replaced with serum-containing DMEM cell culture medium. After 2-3 days of virus infection, 50 μg / ml of hygromycin B was added for resistance screening for 3 days, and the human glioma cell line U87-luc stably expressing luciferase was obtained after detection. This cell line can be expanded as P0 cells for the next step of animal experiment.

[0075] 3) Tumor cell brain orthotopic transplantation surgery:

[0076] After the mouse was anesthetized with isoflurane inhalation anesthesia machine, the animal was fixed to the brain positioning instrument to make the head fixed. The scalp outside the skull was cut longitudinally with a scalpel, and the fontanel point was found. The needle was rotated to pierce the hole with a 5-milliliter syringe needle, and the skull layer was destroyed. 5x10 5 U87-luc cells (P0) were resuspended in 10 μl of normal saline, and the cells were taken up with a 10 μl microsyringe. The injection needle was inserted about 2 mm from the right front temporal side of the nude mouse, and the injection needle was fixed to start injecting the cells at a speed of 1 μl per minute. After injection, the needle was slowly withdrawn, and the skin was sutured. After the animal woke up, it was sent back to the animal house.

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

[0078] 4) After 7 days of successful engraftment of the tumor in the brain of mice, oral treatment of DMSO or Temozolomide (TMZ) was initiated at a dose of 50 mg / kg / day, wherein DMSO was used as a negative control. The treatment regimen was 5 days of treatment per week with 2 days of rest, and the treatment was continued for 3-4 weeks. Six mice were used in each treatment group, and the treatment was repeated three times. The treatment cycle with drug administration-rest was used to mimic the treatment cycle of tumor chemotherapy drugs, so that the process of cell drug resistance was closer to the actual situation in clinic.

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

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

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

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

[0083] Example 2: Basic characteristic analysis of lncATRIG

[0084] 1. RACE (rapid amplification of cDNA ends) 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, first of all, the RNA sequence of lncATRIG and the accurate positioning coordinates on the chromosome are identified by RACE experiment. The experiment mainly uses cDNA amplification kits for 5' and 3' RACE kit (item number: 634858), and the specific steps are carried out according to the kit instructions, which are briefly described as follows:

[0086] (1) Extracting RNA

[0087] First of all, the total RNA is extracted from U87 by using the conventional method of TRIZOL according to the instructions, and it is necessary to ensure that the RNA quality is high and there is no degradation in order to obtain accurate results.

[0088] (2) Reverse transcription to generate cDNA

[0089] 3'RACE: The specific primer containing poly(T) is used for reverse transcription reaction of mRNA with poly(A) tail to generate cDNA with known 3' sequence. Reverse transcriptase reverses mRNA into single-stranded cDNA.

[0090] 5'RACE: Specific gene primer (GSP) is used for reverse transcription to synthesize cDNA from the 3' end of mRNA. Then, through tailing reaction, poly(C) or other specific sequences are added to the 5' end of cDNA, which is convenient for subsequent amplification.

[0091] (3) PCR amplification

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

[0093] 5'RACE: The 5' cDNA with tail is amplified by PCR using universal primer and GSP to obtain the amplification product of 5' end.

[0094] (4) Second round of PCR

[0095] In order to improve the specificity of amplification, it is often necessary to carry out the second round of PCR. 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 products are detected by gel electrophoresis to confirm whether the size of the amplified fragments is as expected. The amplified products can be sequenced after purification to determine the complete 5' and 3' end sequences.

[0098] Example 2 Identification of the basic characteristics of long non-coding RNA lncATRIG obtained and utilized by RNA sequencing (RNA-seq) and transcriptomic analysis, and assembled by bioinformatics methods The full-length cDNA sequence of lncATRIG of 3837 nt was obtained by RACE experiment and Sanger sequencing, and the results are shown in Figures 1A-1B The cDNA sequence of LncATRIG is shown in Seq ID No. 1 (3837 nt):

[0099]

[0100] As Figure 1A The 5' RACE (left) and 3' RACE (right) PCR product agarose gel electrophoresis map of lncATRIG is shown, the transcription start and end sites of lncATRIG are amplified and identified by 5' RACE and 3' RACE methods, the location coordinates and sequence of lncATRIG are shown, and the existence of the full-length transcript is confirmed. The analysis of RACE experiment and previous RNA-seq combined with histone ChIP-seq data determines 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 visualization map of lncATRIG in the gene region of chromosome 6 is shown by the analysis results of transcriptome sequencing and histone antibody chromatin immunoprecipitation DNA sequencing data. From Figure 1B It can be seen that the lncATRIG gene expression peak map analyzed by RNA-seq shows that lncATRIG is significantly up-regulated in temozolomide-resistant glioma cell T3-U87. The transcriptional activity of lncATRIG was verified, and the key histone modification markers were detected, from Figure 1B It can be seen that the lncATRIG gene region has a higher peak overlap with the acetylation of histone H3 on K27 (H3K27ac) and the trimethylation of histone H3 on K4 (H3K4me3) (H3K27ac and H3K4me3 are usually related to enhancers and promoters of active genes), which indicates that this region has an active transcriptional state, and the enhancer and promoter regions of the lncATRIG gene are also defined in the position of the two modification overlap peaks.

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

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

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

[0104] Table 1. PCR primer sequences for hybridization probes

[0105]

[0106] After hybridization, the membrane was treated with SuperBlock containing an RNase inhibitor at room temperature. TM Block with blocking buffer (Thermo Scientific, EO0384) for 1 hour, then use... 800CW streptavidin (Li- The cells were treated with secondary antibody detection reagent (VWR, 102673-342) for 1 hour at room temperature. Finally, imaging was performed using a 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 panel shows the results of Northern blot transfection followed by slide development. This panel demonstrates that the lncATRIG RNA transcript band was detectable in both U87 and drug-resistant T3-U87 cells. Furthermore, band grayscale analysis showed that the expression level of lncATRIG was significantly higher in drug-resistant U87 cells than in primary U87 cells. Signal bands corresponding to 28S and 18S rRNA are also shown. Figure 1C The image on the right shows the RNA electrophoresis results from a Northern blot experiment using formaldehyde-denaturing agarose gel. This image displays the positions of the 28S and 18S rRNA bands after the total RNA from U87 cells (T3-U87 cells, a drug-resistant cell line) was electrophoresed. In RNA electrophoresis, the size of the bands can be roughly determined based on the size of the ribosomal RNA; 28S RNA is typically 5kb, and 18S RNA is typically 2kb. Figure 1CThe right gel electrophoresis map corresponds to show that the RNA band size of lncATRIG is between 28s and 18s, which proves 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) experiment 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 directly observed. The main steps include: fixing the tissue or cells, then hybridizing the fluorescently labeled lncATRIG probe or NEAT1 probe with the target RNA, after washing to remove non-specific binding, using a fluorescence microscope or confocal microscope to observe and image. It is a more common and mature localization technology. The FISH probes of lncATRIG and NEAT1 are synthesized by Beijing Deao Biotechnology Co., Ltd.

[0110] The localization of lncATRIG in cells was detected by smFISH, and the results are shown in Figure 1D Figure 1D The localization image of lncATRIG RNA in human glioma cell lines analyzed by single molecule fluorescence in situ hybridization method is shown in Figure 1D It can be seen from

[0111] From Figures 1A-1D It can be seen that through chromosome localization, histone modification, RACE amplification and cell localization experiments, the basic characteristics of lncATRIG are fully elucidated, which lays a foundation for subsequent research on the function of lncATRIG in temozolomide resistance.

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

[0113] I. Digital PCR (Digital PCR) to detect lncRNA copy number.

[0114] 1. Sample preparation

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

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

[0117] 2. dPCR reaction preparation

[0118] Primer design: Specific primer sequences for target lncRNA were designed as follows:

[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] ACTIN as control group, primer sequence is:

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

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

[0124] 3. Digital partitioning

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

[0126] 4. Result analysis

[0127] By analyzing the fluorescence signal of each unit, dPCR can directly count the number of positive units containing amplification products and the number of negative units without amplification. Calculate the copy number: through the ratio of positive units to total units, and Poisson distribution to calculate the absolute copy number of lncRNA in the initial sample.

[0128] 5. Data analysis

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

[0130] From Figure 2AAs can be seen, there are about 1000 copies of lncATRIG in T3-U87 cells, and 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] II. Detection of lncATRIG expression in normal tissues and glioma tissues by fluorescence quantitative PCR

[0132] 1. Glioma tissues:

[0133] In this embodiment, 15 cases of low-grade glioma (LGG) and 15 cases of glioblastoma (GBM) were used, which were obtained from the surgical resection tissues of patients diagnosed with glioma in Beijing Tiantan Hospital. The informed consent of the patients was obtained before the tissues were obtained, and the patients were informed of the use of the research. The research was 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 cases of LGG and GBM patients, and 2 μg was used for reverse transcription into cDNA for fluorescence quantitative PCR (RT-qPCR) analysis.

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

[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, and the ACTIN upstream primer sequence (and the downstream primer sequence was the same as the primer used in digital PCR. According to the experimental results, qPCR was used for relative quantitative analysis, and after three repeated experiments, the relative expression level statistical chart was obtained, as shown in Figure 2B CCAT1 (a known long non-coding RNA associated with rectal cancer, with high expression abundance in various tumors) was also detected in these glioma tissues as a control, and the upstream primer sequence of CCAT1 (Seq ID No. 16) was 5'-GCCGTGTTAAGCATTGCGAA-3'; the downstream primer sequence was (Seq ID No. 17): 5'-AGAGTAGTGCCTGGCCTAGA-3', and the results were as followsFigure 2C

[0137] From Figure 2B and Figure 2C It can be seen that the expression of lncATRIG in GBM patient samples is higher than that in low-grade glioma (LGG) tissues. In contrast, the high-abundance and oncogenic lncRNA, CCAT1, shows little difference. These results indicate that lncATRIG is associated with TMZ resistance and is up-regulated in GBM (stage IV drug-resistant recurrent glioma) patient samples.

[0138] III. Fluorescence in situ hybridization detection of expression and cellular localization in glioma and normal tissues

[0139] Glioma tissue chip (Cat No: N095Ct01) was purchased from Xi'an Intelligent Biotechnology Co., Ltd. The chip contains 85 cases of glioblastoma and 10 cases of normal brain tissue. The RNA FISH experiment of the glioma tissue chip was carried out according to the following steps:

[0140] 1. Sample preparation: The tissue chip was deparaffinized (if it was paraffin-embedded tissue), then gradient alcohol dehydrated, and finally balanced with PBS.

[0141] 2. Antigen repair: The tissue was subjected to antigen repair by heating or enzyme treatment to improve the entry and binding efficiency of the probe.

[0142] 3. Pretreatment: The tissue was treated with protease to increase the permeability of the tissue, so that the probe can enter the cell.

[0143] 4. Probe hybridization: The specific lncATRIG and NEAT1 probes labeled with fluorescence (the same FISH probes used in Example 1) were added to the sample and hybridized at 37°C overnight.

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

[0145] 6. Staining: The cell nucleus was stained with nuclear dyes such as DAPI for 15 minutes.

[0146] 7. Mounting: The mounting agent (Vector, H1000) was used to mount the sample.

[0147] 8. Microscopic observation: The Zeiss LSM700 laser confocal fluorescence microscope was used for observation and photography, and the results are shown in Figure 2D .

[0148] From Figure 2D ​As can be seen, 90% of GBM tissues are positive for lncATRIG expression and have co-localization with NEAT1 compared with low positive signals in normal brain tissue samples, and lncATRIG is mainly located in the nucleus. As shown in Figure 2, the positive cells in the tissue chip are mainly located in the nucleus. As shown in Figure 3, the statistical results of positive cells in normal brain tissue and GBM tissue have significant differences. Figure 2E

[0149] According to the above results, it can be seen that lncATRIG is determined to be a new long non-coding RNA gene on chromosome 6 by the above RACE, NB and FISH experiments. lncATRIG is highly expressed in 90% of GBM tumor tissues, and the expression has co-localization with NEAT1 (which is a long non-coding RNA that has been reported to be related to tumor drug resistance and high expression), indicating that lncATRIG is similar to NEAT1 in function and also participates in the biological occurrence and drug resistance process of GBM.

[0150] Example 4: Promotion of lncATRIG overexpression on temozolomide resistance

[0151] This example uses the method of overexpressing lncATRIG to verify its effect in glioma cells, especially the influence on temozolomide (TMZ) resistance, and evaluates the cell proliferation ability and invasion ability through colony formation experiment and invasion experiment.

[0152] Experimental method:

[0153] I. Overexpression of lncATRIG in glioma cells

[0154] 1. Cell culture: three human glioma cell lines U87, LN229 and U251 were used, and DMEM medium containing 10% serum was used for culture.

[0155] 2. Plasmid construction

[0156] In order to overexpress lncATRIG, the full-length exon sequence (3,837 nt) was PCR amplified from the cDNA of T3-U87 cells and then cloned into the pLenti-CMV-GFP-Hygro plasmid (Addgene, #656-4). At the same time, a plasmid containing equal length λ DNA 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-Long 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] According to the Lipo2000 transfection reagent instruction, briefly described as follows:

[0166] (1) One day before transfection, U87 cells, LN229 cells or U251 cells were seeded at 3x105cells / well into 6-well plates to ensure that the cells were in the logarithmic growth phase on the day of transfection. 5

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

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

[0169] (4) Place the cells in a 37°C, 5% CO2 incubator, and replace the medium with serum-containing medium after 4-6 hours.

[0170] (5) Observe the state of the cells 24 hours after transfection, and detect the transfection efficiency and gene expression by RT-qPCR method after 48 hours.

[0171] II. Colony formation experiment

[0172] (1) Cell seeding and culture:

[0173] After culturing the U87 cells, LN229 cells or U251 cells overexpressing lncATRIG for 24 hours after transfection, count the cells. Seed 5000 cells per well into a 6-well plate to ensure uniform cell seeding. ​

[0174] (2) Cell treatment and colony formation

[0175] After the cells adhere to the wall, 10 μM TMZ or DMSO is added for treatment. After 48 hours, the culture medium is replaced with fresh drug-free medium, and the cells are cultured for another 10 days. Cell proliferation and visible colonies (a colony usually consists of more than 50 cells) can be observed.

[0176] (3) Fixation of cells

[0177] Methanol or ice-cold methanol: ethanol (1:1) solution is prepared as a fixing solution. The culture medium is discarded, and the cells are gently washed twice with PBS, and then the fixing solution is added. The cells are fixed at room temperature for 15 minutes, and the fixing solution is aspirated, and the plate is kept dry at room temperature.

[0178] (4) Crystal violet staining

[0179] 0.5% crystal violet staining solution is prepared: 0.5 g of crystal violet is dissolved in 100 ml of 20% methanol aqueous solution. 1 ml of crystal violet staining solution is added to the fixed cells, and the cells are stained at room temperature for 15 minutes. The staining solution is discarded, and the plate is rinsed with tap water until the background becomes clear and transparent, and no excess dye remains.

[0180] (5) Drying and observation and photographing

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

[0182] (6) Data analysis

[0183] The number of colonies in each well or dish is counted. The colony formation rate is calculated according to the formula: colony formation rate (%) = number of colonies in the experimental group / number of inoculated cells x 100. The colony formation rates of the treatment group and the control group are compared to evaluate the effect of TMZ on the proliferation ability of the cells.

[0184] The results are shown in Figures 3A-3F The same length (3.8 kb) of λ-DNA and lncATRIG plasmid was overexpressed in U87, LN229 and U251 cells, respectively. The two groups of cells were treated with dimethyl sulfoxide (DMSO, solvent control) and temozolomide (TMZ, chemotherapy drug), respectively. In the three human glioma cell lines U87, LN229 and U251, compared with the cells overexpressing the same length (3.8 kb) of λ-DNA (control cells, Ctrl), lncATRIG overexpression (ATRIG OE) significantly increased the drug resistance of the cells to TMZ. Figures 3A-3FThe results of the colony formation experiment in FIG. 6 show that after TMZ treatment, the cell line overexpressing lncATRIG retained more cell viability and formed more colonies than the control group (Ctrl: λDNA group).

[0185] III. Cell invasion assay (Transwell assay)

[0186] 1. Cell culture:

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

[0188] 2. Transwell chamber preparation: 8 μm pore size chambers were used, and 500 μl of cell culture medium containing 10% serum was added to the lower chamber.

[0189] 3. Cell inoculation:

[0190] Cell counting, adding cell suspension to the upper chamber, usually 1 x 10 5 cells per well, using serum-free medium to prepare cell suspension.

[0191] 4. Place the Transwell chamber in the incubator, usually at 37°C, 5% CO2, 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. Stain with 0.5% crystal violet solution at room temperature for 15 minutes.

[0194] 6. Washing and observation analysis:

[0195] Wash with PBS to remove unbound stain. Observe the cells under a microscope and count the number of migrated and invaded cells. According to the counting results, calculate the number of migrated or invaded cells and perform statistical analysis, the results are shown in Figures 3G-3L .

[0196] As can be seen from Figures 3G-3L , lncATRIG overexpression also enhances the invasion ability of U87 cells, LN229 cells, and U251 cells in the cell invasion experiment, especially under TMZ treatment, the invasion index is significantly increased.

[0197] Example 5: Effect of lncATRIG inhibitor on proliferation and invasion ability 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 glioma cell drug resistance.

[0199] Firstly, according to the RNA sequence of the above lncATRIG gene, an inhibitor targeting degradation of ATRIG, i.e. an ASO sequence modified by locked nucleic acid, is designed. Locked nucleic acid (LNA) is a modified sequence in which one or more of the ribose 2' and 4' carbons are linked together. It is commonly found in A-DNA or RNA. Such nucleic acids can increase the melting temperature (Tm value) of primers or probes, and enhance the stability of these experimental substances. According to the RNA structure of lncATRIG, three antisense oligonucleotides targeting the full length of lncATRIG, Seq ID No. 2-Seq ID No. 4, are designed, each with a length of 16 nt, and locked nucleic acid and methoxy modification on each nucleotide. All sequences are synthesized by QIAGEN (China) Company.

[0200] The sequences of the inhibitors targeting lncATRIG are shown in Table 2:

[0201] Table 2: Sequences of inhibitors targeting lncATRIG

[0202] Name Seq ID No. 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 specific lncATRIG inhibitors were transfected by Lipo, three LNAs were mixed and added to 5x10 5 In T3-U87 cells, the expression level of lncATRIG was detected by RT-qPCR method 48 hours after transfection to determine the knockdown efficiency, as shown in Figure 4A As shown, after transfection of three target LNAs, it is called LNA-ATRIG (LNA-NC is the control), and the total RNA of the cells is extracted to detect the RNA knockdown efficiency of lncATRIG by RT-qPCR. The results show that after LNA-ATRIG transfection, the RNA expression level of lncATRIG in the cells can be reduced by 85-90%.

[0204] The present example then carries out 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: after knocking down lncATRIG, the T3-U87 cells are cultured in a culture medium containing TMZ, and about 1000 cells per well are inoculated into a 6-well plate, 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 ends, the cells are fixed using 4% paraformaldehyde, and the colonies are stained using crystal violet staining method, washed, and observed and counted under a microscope, as shown in Figure 4B . Figure 4C The quantitative analysis results of the number of colonies are shown. From Figure 4B and Figure 4C it can be seen that the T3-U87 cells with knocked down lncATRIG have reduced proliferation ability under the action of TMZ compared with the control group, indicating that knocking down lncATRIG has a recovery 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 the cells is also evaluated by a Transwell experiment. The experimental method is the same as that in Example 3. The T3-U87 cells with knocked down lncATRIG are inoculated into a Transwell chamber containing a basement membrane coating and cultured for 24 hours. TMZ is added to the lower chamber of the Transwell, and after treatment, the uninvaded cells are removed with a cotton swab and stained with crystal violet, and the number of penetrated cells is counted, as shown in Figure 4D . Figure 4E The quantitative analysis statistical results of the invasion index are shown. From Figure 4D and Figure 4E it can be seen that the T3-U87 cells with knocked down lncATRIG have reduced invasion ability under the action of TMZ compared with the control group, indicating that knocking down lncATRIG has a recovery effect on the drug sensitivity of drug-resistant cells, thereby inhibiting the invasion ability of drug-resistant tumor cells.

[0206] The RT-qPCR results show that the expression level of lncATRIG is significantly reduced, verifying the effectiveness of the knockdown. Further analysis of the proliferation and invasion ability of the cells shows that after knocking down lncATRIG, the colony formation ability of the T3-U87 cells under the condition of TMZ treatment is significantly reduced, and the quantitative analysis also confirms 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 ability of TMZ-resistant U87 cells. Knockdown of lncATRIG significantly inhibited the colony formation and invasion ability of cells, suggesting that it can be a potential therapeutic target. By inhibiting the expression of lncATRIG, it can enhance the therapeutic effect of TMZ on glioma and reduce drug resistance. Therefore, intervention measures targeting lncATRIG can help improve the clinical efficacy of TMZ and further explore its potential application in glioma treatment.

[0208] Example 6: lncATRIG inhibitor promotes TMZ-induced DNA damage and apoptosis to increase TMZ sensitivity

[0209] This example investigates whether the lncATRIG inhibitor increases TMZ sensitivity in T3-U87 cells by enhancing TMZ-induced DNA damage and promoting apoptosis.

[0210] The main experimental methods are as follows:

[0211] I. CCK8 calculates the IC50 value of cell response to TMZ after knocking down lncATRIG

[0212] Specific lncATRIG inhibitors were used to transfect T3-U87 cells in order to achieve effective knockdown of lncATRIG. After knockdown, CCK8 method was used to detect the drug resistance of cells to TMZ, and IC50 value was calculated to evaluate the sensitivity of cells to TMZ, as shown in Figure 5A The results showed that the drug half-lethal dose of drug-resistant cells T3-U87 after knocking down lncATRIG decreased, and the drug sensitivity of cells to TMZ significantly improved.

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

[0214] (1) Cell culture:

[0215] TMZ-resistant cells T3-U87 with knockdown of lncATRIG were seeded at 2000 per well in a 96-well plate for culture, and cultured for 24 hours to reach the logarithmic growth phase. U87 cells were used as a control group.

[0216] (2) TMZ treatment:

[0217] Prepare a series of drug solutions with different concentrations (usually in the order of 600 μΜ, 500 μΜ, 400 μΜ, 300 μΜ, 200 μΜ, 100 μΜ from high to low concentration gradient), and add these different concentrations of TMZ-containing medium to different wells of the cell culture plate. Set 3 replicates 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 function.

[0218] (3) CCK8 staining:

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

[0220] (4) Calculate the IC50 value of different cells in response to TMZ according to the OD value:

[0221] According to the OD value under different drug concentrations, draw the curve of TMZ concentration and cell survival rate (ratio of OD value to control group). Through data fitting (for example, using nonlinear regression analysis), calculate the drug concentration that reduces the cell survival rate by 50%, that is, the IC50 value.

[0222] II. Immunofluorescence analysis of the expression changes of DNA damage markers

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

[0224] III. TUNEL method to detect apoptosis after lncATRIG knockdown

[0225] In addition, the effect of lncATRIG knockdown on apoptosis was evaluated by TUNEL staining. In the experiment, T3-U87 cells were subjected to TUNEL staining after 48 hours of TMZ treatment, and the number of TUNEL-positive cells was observed and counted. The right side quantitative analysis shows the results of TUNEL-positive cells after 48 hours of TMZ treatment.

[0226] The results of CCK8 experiment are shown in Figure 5A , and the knockdown of lncATRIG significantly reduced the resistance of T3-U87 cells to TMZ, and the IC50 value decreased significantly. This indicates that lncATRIG is a key regulator of TMZ resistance, and inhibition of its expression can improve the effect of chemotherapy drugs. Further, the results of immunofluorescence experiment are shown in Figure 5B and Figure 5C , and the lncATRIG inhibitor promotes DNA damage accumulation by enhancing the expression of γ-H2AX and RAD51 induced by TMZ. DNA damage is the main mechanism of TMZ-induced cell death, and the knockdown of lncATRIG makes the cells more sensitive to DNA damage, showing its important regulatory role in the DNA repair pathway. At the same time, the results of TUNEL experiment are shown in Figure 5D and Figure 5E , and the knockdown of lncATRIG significantly enhances the apoptosis rate after TMZ treatment. Apoptosis is a self-destruction mechanism of cells when DNA damage is irreparable, and the effect of lncATRIG inhibitor aggravates this process, thereby enhancing the therapeutic effect of TMZ.

[0227] As can be seen from Figures 5A-5E , the lncATRIG inhibitor significantly improves the sensitivity of T3-U87 cells to TMZ by enhancing the DNA damage and apoptosis induced by TMZ. It shows that lncATRIG as an important regulator of TMZ resistance, may become a potential therapeutic target for targeting glioma drug 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 drug-resistant cell mouse brain transplanted tumor to TMZ

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

[0230] Experimental method:

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

[0232] 2. Establishment of TMZ-resistant U87 cell line stably knocked down lncATRIG

[0233] 3. Establishment of intracranial tumor model in nude mice.

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

[0235] 2) Tumor cell brain orthotopic transplantation surgery:

[0236] After anesthesia of the nude mice with isoflurane inhalation anesthesia machine, the animals were fixed to the brain positioning instrument, and the head was fixed immovably. The scalp outside the skull was cut longitudinally with a scalpel, and the bregma point was found, and the needle insertion site was the right front temporal side. A 5-milliliter syringe needle was rotated to make a hole and destroy the skull layer. 5 x 10 5 U87 cells stably expressing firefly luciferase (P0) were resuspended in 10 μl of normal saline, and the cells were taken up with a 10 μl microsyringe. The injection needle was inserted about 2 mm from the right front temporal side of the nude mouse, and the injection needle was fixed to start injecting the cells at a speed of 1 μl per minute. After injection, the needle was slowly withdrawn, and the skin was sutured. After the animals woke up, they were sent back to the animal house.

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

[0238] 3) After successful orthotopic transplantation of tumors in mice, oral administration of DMSO (50 mg / kg / day) or TMZ (50 mg / kg / day) treatment was started 7 days later. The treatment regimen was 5 days of treatment per week, with 2 days of drug withdrawal, lasting for 3-4 weeks. Six mice were taken from each treatment group, and a total of three repeated experiments were performed.

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

[0240] During the treatment, the growth of tumors was monitored using a small animal live imaging system. The imaging results on day 7, 14, 21 and 28 showed that the lncATRIG inhibitor combined with TMZ treatment significantly inhibited the growth of intracranial tumors compared with the control group. The live imaging data showed that the luminescence signal of mouse tumors after lncATRIG knockdown was significantly weakened, indicating that the tumor volume and activity were significantly reduced, as shown in Figure 6A Figure 6B The gray value curve of tumor growth was demonstrated, which proved that the tumor growth dynamics were significantly inhibited. In order to evaluate the overall health of mice in different treatment groups, the body weight changes of mice were recorded every week during the experiment, as shown in Figure 6C Figure 6C It can be seen that there was no significant difference in the body weight changes of mice in each group, indicating that the lncATRIG inhibitor had 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, as shown in Figure 6D Figure 6D It can be seen that the overall survival time of mice in the lncATRIG inhibitor and TMZ combination group was significantly prolonged, and the survival rate was higher than that of the TMZ alone or control group.

[0241] From Figures 6A-6D It can be seen that the lncATRIG inhibitor significantly enhanced the inhibitory effect of TMZ on drug-resistant U87 cell brain transplanted tumors, and did not affect the health status of mice. By combining TMZ and lncATRIG inhibitor, tumor growth was effectively inhibited, and survival rate was significantly improved. This indicates the key role of lncATRIG in glioma drug resistance, and by targeting lncATRIG, it is expected to improve the efficacy of TMZ, providing a new treatment strategy for the clinical treatment of drug-resistant glioma.

[0242] The in vivo experiment of this embodiment proves the significant effect of lncATRIG inhibitor in enhancing the effect of TMZ. This lays a foundation for subsequent clinical transformation, demonstrates the potential of overcoming drug resistance by regulating non-coding RNA, and is expected to bring longer survival and better prognosis for glioma patients.

[0243] ​​​As can be seen from the above embodiments, the lncRNA molecule lncATRIG closely related to the TMZ resistance of glioma is obtained by the in vivo mouse drug resistance screening model and transcriptome sequencing technology, and its high expression in drug-resistant cells and its function of promoting tumor cell proliferation, invasion and drug resistance are verified through a series of experiments. This provides a new molecular target for the study of drug-resistant glioma and reveals a new mechanism of glioma drug resistance. The specific antisense oligonucleotide (ASO) inhibitor targeting the sequence of lncATRIG is designed, which can effectively knock down the expression of lncATRIG, thereby significantly inhibiting the proliferation of glioma cells and enhancing the chemotherapeutic effect of TMZ, especially for drug-resistant glioma patients. This inhibitor provides a precise molecular tool for future individualized treatment plan. 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 induced by chemotherapeutic drugs, and improve the apoptosis rate of tumor cells. This brings new treatment options for patients with recurrent glioma and fills the gap of the lack of effective sensitizers in clinical practice. At the same time, it opens up a new path for targeting lncRNA to treat tumors. Its broad application prospects are not limited to glioma, and in the future, it can also be extended to drug resistance problems in other types of cancer. Through the present application, the inhibitor of lncATRIG is expected to provide new molecular targeted treatment for tumor patients and provide a solid scientific basis for tackling the clinical problem of glioma drug resistance.

[0244] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements can be made and these improvements should also be considered as the protection scope of the present application.

Claims

1. An IncATRIG inhibitor, characterized in that, one or more of the following sequences LNA-1, LNA-2, LNA-3; wherein the sequence of LNA-1 is shown as Seq ID No. 2; the sequence of LNA-2 is shown as Seq ID No. 3; the sequence of LNA-3 is shown as Seq ID No. 4; the cDNA sequence of the lncATRIG is shown as Seq ID No. 1; the LNA-1, the LNA-2 and the LNA-3 are each modified with a locked nucleic acid and a methoxy modification on each nucleotide.

2. Use of the inhibitor of lncATRIG according to claim 1 in the preparation of a medicament for inhibiting proliferation of brain glioma cells.

3. Use of the inhibitor of lncATRIG according to claim 1 in the preparation of a medicament for temozolomide sensitization of brain glioma.

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