LncRNA WARS2-IT1 inhibitors and their applications

Abstract

The present invention relates to biomedicine, and in particular to LncRNA WARS2-IT1 inhibitors and applications thereof. After knocking down or silencing LncRNA WARS2-IT1 using the LncRNA WARS2-IT1 inhibitor, the proliferation ability, migration ability, and invasion ability of glioma cells are significantly reduced. The LncRNA WARS2-IT1 inhibitor can be used in the preparation of products such as diagnosis and treatment of gliomas and improving the sensitivity of targeted drugs, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a LncRNA WARS2-IT1 inhibitor and an application thereof. Background Art

[0002] Temozolomide (TMZ) is the only first-line chemotherapy drug for the treatment of gliomas, especially in the treatment of malignant gliomas after surgical resection. It can effectively improve the overall survival rate of patients due to its good blood-brain barrier permeability and significant efficacy. However, a considerable number of patients still show primary or secondary resistance to TMZ, leading to chemotherapy failure. It is urgent and necessary to conduct in-depth research on the mechanism of temozolomide resistance and to find potential biomarkers that can predict the effect of targeted therapy. Summary of the invention

[0003] Based on this, the present invention provides a LncRNA WARS2-IT1 inhibitor and application thereof, which at least solves one problem in the prior art.

[0004] In a first aspect, the present invention provides a LncRNA WARS2-IT1 inhibitor, wherein the LncRNA WARS2-IT1 inhibitor is si-WARS2-IT1-#1 or si-WARS2-IT1-#2, the target sequence of the si-WARS2-IT1-#1 is CCTGGAGCAAAGGTGACTT, and the target sequence of the si-WARS2-IT1-#2 is CTCCACGAAGCTGAGTATT.

[0005] The sequence of LncRNA WARS2-IT1 has been made public at https: / / www.ncbi.nlm.nih.gov / nuccore / NR_126447.1, specifically: (SEQ ID NO: 1). After knocking down / silencing LncRNA WARS2-IT1, the temozolomide drug sensitivity of glioma cells was significantly increased. CCTGGAGCAAAGGTGACTT (SEQ ID NO: 2) and CTCCACGAAGCTGAGTATT (SEQ ID NO: 3) in LncRNA WARS2-IT1 can be used as new therapeutic molecular markers and drug targets for the treatment of glioma, thereby guiding clinicians to intervene and treat, improve the survival rate and quality of life of patients, and have great application prospects.

[0006] In a second aspect, the present invention provides use of the LncRNA WARS2-IT1 inhibitor in the preparation of a drug for treating or preventing glioma, wherein the drug for treating or preventing glioma contains temozolomide.

[0007] Using the LncRNA WARS2-IT1 inhibitor to interfere with LncRNA WARS2-IT1 can overcome the temozolomide resistance of glioma cells and increase the drug sensitivity of glioma cells to temozolomide, thereby improving the effect of treating or preventing gliomas.

[0008] In a third aspect, the present invention provides a drug for treating or preventing glioma, comprising the LncRNA WARS2-IT1 inhibitor and temozolomide.

[0009] In some preferred embodiments, the dosage form of the drug for treating or preventing glioma is a solution.

[0010] In some preferred embodiments, the solvent of the solution is water.

[0011] In some preferred embodiments, the LncRNA WARS2-IT1 inhibitor is used at a concentration of 100 nM, and the temozolomide is used at a concentration of 400 μM.

[0012] In a fourth aspect, the present invention provides a reagent for detecting LncRNA WARS2-IT1, comprising a primer pair with detection specificity for LncRNA WARS2-IT1, wherein the sequence of the forward primer of the primer pair is 5′-CGAGGCCCTTATGGAGAACC-3′ (SEQ ID NO: 4), and the sequence of the reverse primer of the primer pair is 3′-TCATGCCATCCCAACAGTCC-5′ (SEQ ID NO: 5).

[0013] In a fifth aspect, the present invention provides a glioma diagnosis and evaluation kit, which includes the reagent for detecting LncRNA WARS2-IT1.

[0014] In some preferred embodiments, the kit further comprises TB green, ddH2O and a cDNA template.

[0015] In some preferred embodiments, the concentrations of the forward primer of the primer pair and the reverse primer of the primer pair are both 10 μM. For example, if the reaction system of the kit is 20 μL, it may contain 10 μL of TB green, 0.8 μL of the forward primer of the primer pair, 0.8 μL of the reverse primer of the primer pair, 6.4 μL of ddH2O, and 2 μL of cDNA template.

[0016] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects:

[0017] After knocking down or silencing LncRNA WARS2-IT1 using the LncRNA WARS2-IT1 inhibitor, the proliferation ability, migration ability, and invasion ability of glioma cells are significantly reduced. The LncRNA WARS2-IT1 inhibitor can be used in the preparation of products such as the diagnosis and treatment of gliomas and improving the sensitivity of targeted drugs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Scatter plot of the expression profile data of lncRNA WARS2-IT1 in glioma and normal brain tissues in the TCGA-GTEx database for bioinformatics analysis.

[0019] Figure 2 This is a graph showing the expression of lncRNA WARS2-IT1 in glioma tissue and normal brain tissue.

[0020] Figure 3 The ROC curve reflecting the relationship between specificity and sensitivity of lncRNA WARS2-IT1 is shown.

[0021] Figure 4 This is a correlation analysis chart between high expression of lncRNA WARS2-IT1 and the overall survival of glioma patients.

[0022] Figure 5 This is a correlation analysis chart between high expression of lncRNA WARS2-IT1 and the progression-free interval of prognosis in glioma patients.

[0023] Figure 6 This is a correlation analysis chart between high expression of lncRNA WARS2-IT1 and the prognosis of glioma patients with disease-specific survival.

[0024] Figure 7 This figure shows the effect of interfering with lncRNA WARS2-IT1 on the RNA expression of lncRNA WARS2-IT1.

[0025] Figure 8 This is a graph showing the effect of interfering with lncRNA WARS2-IT1 on the temozolomide drug sensitivity-proliferation ability of glioma T98G and U251 cells.

[0026] Fig. 9 This is a graph showing the effect of interfering with lncRNA WARS2-IT1 on the temozolomide drug sensitivity and apoptosis of glioma T98G and U251 cells.

[0027] Fig.10This is a graph showing the effect of interfering with lncRNA WARS2-IT1 on the temozolomide drug sensitivity-migration ability of glioma T98G and U251 cells.

[0028] Fig.11 This is a statistical analysis of the effects of interfering with lncRNA WARS2-IT1 on the temozolomide drug sensitivity-migration ability of glioma T98G and U251 cells.

[0029] Fig.12 This is a graph showing the effect of interfering with lncRNA WARS2-IT1 on the temozolomide drug sensitivity-invasive ability of glioma T98G and U251 cells.

[0030] Fig.13 This is a statistical analysis of the effects of interfering with lncRNA WARS2-IT1 on the temozolomide drug sensitivity-invasive ability of glioma T98G and U251 cells.

[0031] Fig.14 Photo of mice used in the experiment on the therapeutic effect of interfering with lncRNA WARS2-IT1 on glioma in vivo.

[0032] Fig.15 Photo of mouse tumors from an experiment on the therapeutic effect of interfering with lncRNA WARS2-IT1 on glioma in vivo.

[0033] Fig.16 This is a graph showing the changes in mouse tumor volume in an experiment on the therapeutic effect of interfering with lncRNA WARS2-IT1 on glioma in vivo.

[0034] Fig.17 This is a graph showing the changes in mouse tumor weight in an experiment on the therapeutic effect of interfering with lncRNA WARS2-IT1 on glioma in vivo. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effect of the present invention.

[0036] Through cell and animal experiments, it was confirmed that si-WARS2-IT1-#1 and si-WARS2-IT1-#2 can target and inhibit the expression of LncRNA WARS2-IT1 gene, and significantly improve the drug sensitivity of temozolomide by inhibiting the proliferation and metastasis of glioma cells and promoting apoptosis.

[0037] 1. Expression of lncRNA WARS2-IT1 in glioma tissue and normal brain tissue

[0038] 1. Analysis of the expression of lncRNA WARS2-IT1 in glioma and normal brain tissues using the TCGA-GTEx database

[0039] To explore the expression and clinical significance of lncRNA WARS2-IT1 in glioma, the expression profile data of lncRNA in glioma and normal brain tissues (689 cases of glioma tissue data and 1157 cases of normal brain tissue data) in the TCGA-GTEx database were analyzed by bioinformatics, and a scatter plot was drawn.

[0040] The results are as follows Figure 1 As shown in Figure 2, lncRNA WARS2-IT1 is significantly more expressed in glioma than in normal brain tissue ( p <0.001), abnormal expression of lncRNA WARS2-IT1 has a certain predictive value for the early diagnosis of glioma patients.

[0041] 2. Clinical sample validation

[0042] The RT-qPCR method was used to detect the expression of lncRNA WARS2-IT1 in glioma and normal brain tissue samples collected by Jiangxi Cancer Hospital (58 glioma tissue samples and 17 normal brain trauma tissue samples). The glioma and brain trauma samples used have passed the medical ethics review of the scientific research project of Jiangxi Cancer Hospital (No. 2020ky008). The detection steps are as follows:

[0043] (1) RNA extraction

[0044] Grind the tissue sample with a tissue grinder at low temperature to avoid cross contamination, and add 1 mL Trizol to each well. Use a pipette to blow repeatedly and let it stand at room temperature for 5 min. Add 200 uL of chloroform to each well, shake and mix on a shaker for 15 s, and then let it stand at room temperature for 2-3 minutes. Centrifuge at 12000 g for 15 min at 4 ℃. Collect the upper aqueous phase with a pipette into a new 1.5 mL centrifuge tube without RNase enzyme. Add an equal volume of isopropanol to the aqueous phase to the centrifuge tube, slowly invert and mix, and let it stand at room temperature for 5-10 min. Centrifuge at 12000 g in a 4℃ centrifuge for 10 min, and RNA will precipitate at the bottom or wall of the tube. Discard the supernatant, pre-cool 75% ethanol prepared with DEPC water (no RNase), add 500 uL to the EP tube to dissolve the RNA, blow and mix thoroughly, and wash the precipitate. Centrifuge at 12000 g for 5 min at 4 ℃. Discard the supernatant and let the EP tube stand in the clean bench for 15-30 min. Add 20 uL of DEPC water to dissolve RNA.

[0045] (2) Reverse transcription reaction

[0046] System 1 (removal of genomic DNA) includes 1 μl gDNA remover, 2 μl 5×gDNA Era buffer, 1 μg total RNA, and RNase-free dH2O to 10 μl; reaction conditions: 42°C, 2 min, 4°C, +∞.

[0047] System 2 (reverse transcription) includes 1 μl Primescript RT enzyme mix, 4 μl 5× Primescript buffer 2, 1 μl RT primer mix, and add RNase-free dH2O to 4 μl.

[0048] After the reaction, add system 1 directly into system 2 and mix well. The reaction conditions are: 37°C, 15 min; 85°C, 5 s; 4°C, +∞.

[0049] (3) Primer design

[0050] According to the sequences of lncRNA WARS2-IT1 and GAPDH gene in NCBI, RT-qPCR amplification primers were designed. The specific primer sequences are as follows:

[0051] lncRNA WARS2-IT1 gene:

[0052] The forward primer was: F: 5′-CGAGGCCCTTATGGAGAACC-3′ (SEQ ID NO: 4);

[0053] The reverse primer was: R: 3′-TCATGCCATCCCAACAGTCC -5′ (SEQ ID NO: 5);

[0054] GAPDH gene:

[0055] The forward primer was: F: 5′-CCCATCACCATCTTCCAGGAG-3′ (SEQ ID NO: 6);

[0056] The reverse primer is: R: 3′-CCCATGTAGCAGACTCCTACTCA-5′ (SEQ ID NO: 7).

[0057] (4) RT-qPCR amplification test

[0058] Mix the cDNA obtained by reverse transcription reaction and pipette 2 μl to prepare the reaction system as follows: 10 μl TB green, 0.8 μl upstream primer, 0.8 μl downstream primer, 6.4 μl ddH2O, 2 μl cDNA.

[0059] The reaction conditions used a two-step PCR standard amplification procedure:

[0060] Step 1: denaturation at 95°C for 30 seconds;

[0061] Step 2: PCR reaction at 95°C for 5s, 60°C for 30s; 40 cycles;

[0062] Step 3: Melting curve: 95℃ 10s, 65℃ 5s.

[0063] The primer concentration was adjusted to 10 μM, GAPDH was used as the internal reference, and the -ΔCt The relative expression level was calculated.

[0064] (5) Results

[0065] The results are as follows Figure 2 As shown, lncRNA WARS2-IT1 expression was significantly increased in glioma tissues ( p <0.05), which is consistent with the results of the TCGA-GTEx database analysis. Therefore, the expression of lncRNA WARS2-IT1 in glioma is significantly higher than that in normal brain tissue, and it may be used as a biomarker for glioma diagnosis.

[0066] In addition, if Figure 3 As shown in the figure, the analysis found that the area under the ROC curve was 0.943, which means that lncRNA WARS2-IT1 may be a potential diagnostic marker for glioma.

[0067] 2. Correlation analysis between high expression of lncRNA WARS2-IT1 and prognosis of glioma patients

[0068] To explore the correlation between lncRNA WARS2-IT1 and the prognosis of glioma patients, including overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI), glioma patients were divided into lncRNA WARS2-IT1 high expression group and low expression group according to the median value, and Cox regression was used for analysis.

[0069] like Figure 4-Figure 6 As shown in the figure, the analysis results showed that the prognosis of patients with high expression of lncRNA WARS2-IT1 was worse than that of glioma patients with low expression, OS: HR = 2.08 (1.59-2.72) P < 0.001, DSS: HR = 2.10 (1.59-2.79) P < 0.001, PFI: HR = 1.98 (1.57-2.50) P < 0.001; HR represents hazard ratio, Figure 4-Figure 6The numbers in the two rows at the bottom of the middle represent the number of glioma patients in the low-expression group and the high-expression group of lncRNA WARS2-IT1, respectively. The above results show that lncRNA WARS2-IT1 has potential application value in the early diagnosis and prognosis survival prediction of glioma patients.

[0070] 3. Determination of the interference efficiency of the lncRNA WARS2-IT1 interference fragment

[0071] 1. siRNA transfection

[0072] lncRNA WARS2-IT1 siRNA target sequence: The target sequence of si-WARS2-IT1-#1 is CCTGGAGCAAAGGTGACTT (SEQ ID NO: 2), and the target sequence of si-WARS2-IT1-#2 is CTCCACGAAGCTGAGTATT (SEQ ID NO: 3).

[0073] Glioma cells U251 and T98G were seeded in 6-well plates and transfected when the cells grew to a confluency of 50-60%. First, discard the old cell culture medium with a Pasteur pipette, gently wash the cells 1-2 times with PBS, and add 1.5 mL of DMEM medium containing 10% FBS to each well. Prepare the transfection system: take 4 sterile enzyme-free 1.5 mL EP tubes, add 250 μL Opti-MEM to each, add 10 μL si-WARS2-IT1-#1, si-WARS2-IT1-#2, and NC fragment (an RNA fragment that will not knock down any other genes), and gently pipette to mix and let stand for 5 minutes; take another 4 1.5 mL EP tubes, add 250 μL Opti-MEM to each, and then add 8 μL RNA iMAX to each, and gently pipette to mix and let stand for 5 minutes. The two systems were mixed, allowed to stand for 20 min, and then added to the wells of the corresponding cells. The cells were placed in a cell culture incubator and cultured for 48 h.

[0074] 2. RNA Extraction

[0075] Take the glioma cells U251 and T98G that have been transfected for 48 hours, discard the old cell culture medium with a Pasteur pipette, gently wash the cells 1-2 times with PBS, and add 1 mL Trizol to each well. Use a pipette to blow repeatedly and let it stand at room temperature for 5 min. Add 200 uL of chloroform to each well, shake and mix on an oscillator for 15 s, and then let it stand at room temperature for 2-3 min. Centrifuge at 12000 g for 15 min at 4 ℃. Collect the upper aqueous phase with a pipette into a new 1.5 mL centrifuge tube without RNase enzyme. Add an equal volume of isopropanol to the centrifuge tube and mix it upside down slowly, and let it stand at room temperature for 5-10 min. Centrifuge at 12000 g in a 4 ℃ centrifuge for 10 min, and the RNA will precipitate at the bottom or wall of the tube. Discard the supernatant, precool 75% ethanol prepared with DEPC water, add 500 uL to the EP tube to dissolve the RNA, blow and mix thoroughly, and wash the precipitate. Centrifuge at 4°C, 12000 g for 5 min. Discard the supernatant and place the EP tube in a clean bench for 15-30 min. Add 20 uL of DEPC water to dissolve the RNA.

[0076] 3. RNA Concentration Determination and Dilution

[0077] Adjust the sample RNA concentration between 200-1000 ng / uL (if the RNA concentration is too high, dilute it with DEPC water).

[0078] 4. RT-qPCR detection

[0079] The relative expression of lncRNA WARS2-IT1 was measured, and the interference efficiency of si-WARS2-IT1-#1, si-WARS2-IT1-#2 and si-NC groups was calculated.

[0080] 5. Results

[0081] The results are as follows Figure 7 As shown, si-WARS2-IT1-#1 and si-WARS2-IT1-#2 both had good knockdown efficiency of lncRNA WARS2-IT1 in glioma T98G and U251 cells.

[0082] Effects of interfering with lncRNA WARS2-IT1 on temozolomide sensitivity, cell proliferation and apoptosis in glioma T98G and U251 cells

[0083] 1. MTS experiment to detect the effect of interfering with lncRNA WARS2-IT1 on temozolomide drug sensitivity and cell proliferation ability of glioma U251 and T98G cells

[0084] After the glioma U251 and T98G cells grew to the logarithmic growth phase, they were plated in 96-well plates. The glioma cells were first grouped into si-NC, si-WARS2-IT1-#1, si-NC+TMZ, and si-WARS2-IT1-#1+TMZ groups. According to the grouping information, the lncRNA WARS2-IT1 interference fragment siRNA and NC were transfected into the glioma cells according to the transfection steps. After being cultured in a constant temperature cell culture incubator for 24 h, the latter two groups were added with a culture medium containing 400 μM temozolomide, and the culture was continued for 72 h, and the cell survival rate of each group was detected.

[0085] like Figure 8 As shown in the results, in glioma U251 and T98G cells, the cell proliferation ability of the si-WARS2-IT1-#1+TMZ group was significantly lower than that of the si-WARS2-IT1-#1 group; at the same time, after interfering with lncRNA WARS2-IT1, the activity rate of the si-WARS2-IT1-#1+TMZ group was significantly lower than that of the si-NC+TMZ group, and the cell activity survival rate of the si-WARS2-IT1-#1+TMZ group was significantly lower than that of the si-WARS2-IT1-#1 group. The above results show that the survival rate of glioma cells in the group with high expression of lncRNA WARS2-IT1 increased, and the drug sensitivity was low; interfering with lncRNA WARS2-IT1 can significantly increase the drug sensitivity of TMZ. At the same time, silencing lncRNA WARS2-IT1 has the potential value of overcoming temozolomide resistance and causing the tumor to continue to progress, providing new targeted therapeutic drugs for the clinical treatment of glioma.

[0086] 2. Flow cytometry to detect apoptosis and analyze the effect of interfering with lncRNA WARS2-IT1 on temozolomide sensitivity and apoptosis in glioma U251 and T98G cells

[0087] Glioma U251 and T98G cells in the logarithmic growth phase were taken and inoculated into six-well plates. When the cells grew to a confluence of 50%-60%, the glioma cells were first divided into si-NC, si-WARS2-IT1-#1, si-NC+TMZ, and si-WARS2-IT1-#1+TMZ groups. According to the grouping information, lncRNA WARS2-IT1 interference fragment siRNA and NC were transfected into glioma cells, respectively. After 12 h of transfection, the culture medium containing temozolomide was added to the latter two groups according to the grouping information and cultured for a further 72 h. The supernatant was collected and centrifuged, washed twice with PBS, digested with trypsin and the collected cells were transferred to the EP tube with the collected supernatant cells, centrifuged at 3000 rpm for 10 min, and the supernatant was discarded; after washing with PBS, 195 μL of binding solution was added to each sample to resuspend the cells, and 5 μL of AnnexinV-FITC and 10 μL of propidium iodide were added in sequence, mixed gently, incubated at room temperature in the dark for 15 min, and cell apoptosis was detected by flow cytometry.

[0088] like Fig. 9 As shown in the figure, the number of apoptotic cells in the si-WARS2-IT1-#1 group was significantly lower than that in the si-WARS2-IT1-#1+TMZ group in glioma U251 and T98G cells; after interfering with lncRNA WARS2-IT1, the number of apoptotic cells in the si-WARS2-IT1-#1+TMZ group of glioma U251 and T98G cells increased significantly compared with si-NC+TMZ. The above results suggest that highly expressed lncRNA WARS2-IT1 can significantly inhibit the sensitivity of TMZ drugs, and reducing the expression of lncRNA WARS2-IT1 can significantly improve the temozolomide drug sensitivity of glioma cells, increase the number of apoptotic cells, and reduce the survival rate. In addition, the above results also show that silencing lncRNA WARS2-IT1 increases the drug sensitivity of temozolomide, and the proportion of apoptotic cells increases significantly, which has the potential value of overcoming temozolomide resistance and provides new targeted therapeutic drugs for the clinical treatment of glioma.

[0089] 5. Effect of interference with lncRNA WARS2-IT1 on temozolomide sensitivity, migration and invasion of glioma U251 and T98G cells

[0090] 1. Scratch assay to detect the effect of interfering with lncRNA WARS2-IT1 on the sensitivity and migration ability of temozolomide in glioma U251 and T98G cells

[0091] 1×10 6 / ml of U251 and T98G cells were added to 6-well plates. Glioma cells were divided into si-NC, si-WARS2-IT1-#1, si-NC+TMZ, and si-WARS2-IT1-#1+TMZ groups. According to the grouping information, lncRNA WARS2-IT1 interference fragment siRNA and NC were transfected into glioma cells respectively. When the cell confluence reached 70%, they were incubated in an incubator for 24 hours. A 1mL pipette tip was used to form horizontal scratches on the bottom surface of the adherent cells. Wash twice in phosphate-buffered saline (PBS) and incubate in an incubator for 24 hours. Wound healing was recorded under an inverted microscope. ImageJ was used to perform statistical analysis of the wound healing area.

[0092] like Fig.10 and Fig.11 As shown in the results, after interfering with lncRNA WARS2-IT1, the healing area of ​​U251 and T98G cells in the si-NC+TMZ group was smaller than that in the si-WARS2-IT1-#1+TMZ group, indicating that the cell migration ability was reduced, revealing that interfering with lncRNA WARS2-IT1 significantly increased the drug sensitivity of glioma T98G and U251 cells to temozolomide treatment by inhibiting the migration ability of glioma cells; at the same time, silencing lncRNA WARS2-IT1 has the potential to overcome temozolomide resistance and cause tumor metastasis, providing new targeted therapeutic drugs for the clinical treatment of glioma.

[0093] 2. Transwell assay to detect the effect of interfering with lncRNA WARS2-IT1 on the sensitivity and invasion ability of temozolomide in glioma U251 and T98G cells

[0094] Glioma cells U251 and T98G growing in logarithmic growth were inoculated in six-well plates and cultured until the confluence was 40-50%. Glioma cells were divided into si-NC, si-WARS2-IT1-#1, si-NC+TMZ, and si-WARS2-IT1-#1+TMZ groups. According to the grouping information, lncRNA WARS2-IT1 interference fragment siRNA and NC were transfected into glioma cells respectively. After the cells were cultured for 48 hours, the cells were digested. Matrigel was prepared: serum-free DMEM containing TMZ (1:8). According to the grouping information, 60 μL of matrigel suspension per well was inoculated into the upper chamber of a 24-well plate with a chamber, and 20% serum DMEM was added to the lower chamber, and the chamber was placed in an incubator for 48 hours. The membrane of the chamber was stained with crystal violet, and the number of invaded cells was observed under a microscope. Finally, the number was counted using Imag J software.

[0095] like Fig.12 and Fig.13 As shown in the figure, after interfering with lncRNA WARS2-IT1, the number of U251 and T98G cell invasions in the si-NC+TMZ group was significantly reduced compared with the si-WARS2-IT1-#1+TMZ group, indicating that the invasive ability of glioma cells was reduced. It was further shown that interfering with lncRNA WARS2-IT1 could increase the drug sensitivity of TMZ in U251 and T98G glioma cells, indicating that lncRNA WARS2-IT1 plays an important role in drug resistance in glioma; at the same time, silencing lncRNA WARS2-IT1 has the potential to overcome temozolomide resistance and cause tumor invasion, providing new targeted therapeutic drugs for the clinical treatment of glioma.

[0096] 6. Detecting the effect of lncRNA WARS2-IT1 on TMZ sensitivity in vivo at the animal level

[0097] All nude mouse tumorigenesis experiments were approved by the animal experiment ethics review of Nanchang Leyou Biotechnology Co., Ltd. (No. RYE2024022602). A mouse glioma xenograft model was established, and human U87-MG cells were grouped and transfected with si-NC and si-WARS2-IT1-#1 (siRNA for animals synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.), each at 200 μL / 1×10 7 The number of cells was subcutaneously inoculated into the scapula of BALB / c nude mice (6 weeks old, female), and the nude mice were randomly divided into four groups: si-NC, si-WARS2-IT1-#1, si-NC+TMZ, and si-WARS2-IT1-#1+TMZ, with 5 mice in each group, in an SPF-grade sterile animal breeding room. The animals were given siRNA at a dose of 2.5 nmol / mouse / day, 3 times a week, for 2 consecutive weeks of intratumoral injection, and TMZ was intraperitoneally injected at a dose of 50 ug / g / day, 5 times a week, for 2 consecutive weeks. Tumor volume was measured with a vernier caliper. After about 14 days, all mice were euthanized by CO2, and the tumor masses were removed to record the tumor size and tumor, and the growth curve was drawn.

[0098] Through continuous monitoring of the mouse xenograft model, it was found that the tumor volume of mice in the si-WARS2-IT1-#1 group was significantly smaller than that in the si-NC group, and the tumor volume of mice in the si-WARS2-IT1-#1+TMZ group was significantly smaller than that in the si-NC+TMZ group ( Fig.14 and Fig.15 In addition, the tumor size and average tumor weight of the TMZ-treated mice group after silencing lncRNA WARS2-IT1 were lower than those of the si-NC+TMZ mice group on day 14 ( Fig.16 and Fig.17 ). The above results show that silencing lncRNA WARS2-IT1 can also increase the drug sensitivity of TMZ in in vivo experiments, further confirming that silencing lncRNA WARS2-IT1 plays an important role in improving the temozolomide treatment of glioma; at the same time, silencing lncRNA WARS2-IT1 has the potential value of overcoming temozolomide (TMZ) resistance, providing new targeted therapeutic drugs for the clinical treatment of glioma.

[0099] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same or equivalent means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. Use of a lncRNA WARS2-IT1 inhibitor in the preparation of a drug for treating glioma, wherein the drug for treating glioma contains temozolomide, the lncRNA WARS2-IT1 inhibitor is si-WARS2-IT1-#1, and the target sequence of the si-WARS2-IT1-#1 is CCTGGAGCAAAGGTGACTT; the lncRNA WARS2-IT1 inhibitor can improve the drug sensitivity of temozolomide.

2. A drug for treating glioma, characterized in that: The invention comprises a LncRNA WARS2-IT1 inhibitor and temozolomide, wherein the LncRNA WARS2-IT1 inhibitor is si-WARS2-IT1-#1, and the target sequence of the si-WARS2-IT1-#1 is CCTGGAGCAAAGGTGACTT; and the LncRNA WARS2-IT1 inhibitor can improve the drug sensitivity of temozolomide.

3. The drug according to claim 2, characterized in that The dosage form of the drug for treating glioma is a solution.

4. The drug according to claim 3, characterized in that The solvent of the solution is water.

5. The drug according to claim 3, characterized in that The LncRNA WARS2-IT1 inhibitor was used at a concentration of 100 nM, and the temozolomide was used at a concentration of 400 μM.

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